An in vitro Method to Test the Safety and Efficacy of Low-Level Laser. Therapy (LLLT) in the Healing of a Canine Skin Model

Transcription

1 An in vitro Method to Test the Safety and Efficacy of Low-Level Laser Therapy (LLLT) in the Healing of a Canine Skin Model by Dominique Gagnon A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Veterinary Science in Department of Clinical Studies Guelph, Ontario, Canada Dominique Gagnon, July, 2015

2 ABSTRACT AN IN VITRO METHOD TO TEST THE SAFETY AND EFFICACY OF LOW-LEVEL LASER THERAPY (LLLT) IN THE HEALING OF A CANINE SKIN MODEL Dominique Gagnon University of Guelph, 2015 Advisor: Dr. Thomas Gibson Low-level laser therapy (LLLT) aims to photoactivate cellular mechanisms that have the potential to improve healing of the treated regions, and reduce local edema, inflammation and pain. Low-level laser therapy has been used clinically as a treatment modality in humans for a variety of medical conditions, including the improvement of wound-healing processes. The results obtained appear to be closely related to parameters such as dose, time of exposure and wavelength. Although LLLT is gaining popularity, a universally accepted theory that explains the cellular effects and beneficial biological processes seen in wound healing has not been described. This research was designed to primarily evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK), in an in vitro wound healing model. A secondary objective was to perform a pilot study with this model in order to evaluate the effect of LLLT on the expression of microrna-21 (mir-21), a microrna associated with wound healing, using quantitative real time polymerase chain reaction (qrt-pcr). Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. The proliferation assay was performed using

3 water-soluble tetrazolium (WST-1), a proliferation reagent. Canine keratinocytes were cultured in tissue culture flasks and then seeded in 6-well tissue culture plates at different cellular concentrations. Cells between the third- and fifth-passage were used for all experiments. Laser treatment of monolayer cultures was performed using a Helium-Neon Class IV laser system. Sham irradiation and different energy doses of 0.1, 0.2, 1.2 and 10 J/cm 2 were evaluated. In this in vitro wound healing model, keratinocytes exposed to doses of 0.1, 0.2, and 1.2 J/cm 2 migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < ) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm 2. Irradiation with 10 J/cm 2 was characterized by decreased cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior and high doses may be detrimental to wound healing.

4 ACKNOWLEDGEMENTS Over the past 3 years of my Doctorate of Veterinary Science (DVSc) and surgery residency training, numerous individuals have supported me and made this challenging adventure incredible. I would like to begin with a special thank you to my DVSc advisor, Dr. Thomas W. Gibson. Thank you for your constant moral support and enthusiasm in the achievement of my thesis-research project and residency training. You were an integral part to the success of this journey and I will always be greatful. I am blessed to have you as an advisor. I would like to thank Dr. Jonathan LaMarre for your expertise, guidance and support through all the laboratory experiment process. You were a great source of encouragement. I would like to extend my sincere gratitude to the other members of my advisory committee, Dr. Ameet Singh and Dr. Alexander zur Linden for your invaluable guidance and support throughout this graduate research project. I would also like to thank Dr. Bryden Stanley for taking part in my DVSc Examination Committee. Thank you as well to Jaimie Kazienko and Thaiza Tiger Costa for your friendship and all your hard work while collecting the experimental data. Much gratitude girls for your ever present positive encouraging attitude. Thank you to William Sears, for your wonderful assistance with the statistical analysis of my project, and all the technical support received while working at the laboratory, including Ed Reyes and Dr. Monica Antenos; you have all been so welcoming and helpful. Thank you so much. iv

5 I would also like to thank my family, René Gagnon, Danielle Gagnon and Marc- Antoine Gagnon, and all my friends who have been so supportive over the years. I am blessed to have such great, carrying and encouraging people in my life! Thank you. Finally, I would like to thank the Ontario Veterinary College Pet Trust Fund for your generous financial support and, LiteCure Companion Therapy Laser System, for providing me with great equipment support. v

6 DECLARATION OF THE WORK PERFORMED I declare that, with the exception of the items below, all work in this thesis was performed by me, Dominique Gagnon. With my guidance, direction and supervision, Jaimie Kazienko and Thaiza Tiger Costa helped with performing the scratch migration assay and proliferation assay. Jaimie Kazienko designed and performed all procedures associated with the microrna (mirna or mir) gene expression experiments, including interpretation of the quantitative reverse transcription polymerase chain reaction (qrt-pcr) used to analyze mir-21 gene expression level. She also wrote the materials and methods for this section. The statistical analysis was performed with the guidance of Williams Sears of the Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario. vi

7 TABLE OF CONTENTS Abstract Acknowledgements Declaration of Work Performed Table of Contents Lists of Tables List of Figures List of abbreviations Page ii iv vi vii x xi xiii CHAPTER I: Literature Review 1.1 Physiology of Wound Healing Phases of wound healing 3 i) Hemostasis 3 ii) Inflammation 5 iii) Proliferation 7 iv) Remodeling or maturation History of Light Therapy Laser Basics Monochromaticity Coherence Collimation Laser Physics Wavelength Energy Power Spot size Power density Continuous and pulsed emission Laser classification Low-Level Laser Therapy Suggested cellular and tissue mechanisms of laser therapy Biphasic dose-response of light therapy The biological effects of laser therapy 23 i) Tisse repair 25 ii) Pain relief 28 iii) Inflammation 30 vii

8 1.6 Laser Safety and Special Considerations MicroRNA Expression MicroRNA biogenesis and function MicroRNA in skin morphogenesis MicroRNA in wound healing MicroRNA and laser therapy Rationale, Hypothesis and Objectives References 37 CHAPTER II: Introduction to Cell Culture Experiments 2.1 Scratch Migration and Proliferation Assays Pilot Study References 47 CHAPTER III: An In Vitro Method to Test the Safety and Efficacy of Low-Level Laser Therapy (LLLT) in the Healing of a Canine Skin Model 3.1 Abstract Background Materials and Methods Cell culture Study protocols Experimental design Low-level laser therapy Scratch migration assay Proliferation assay Statistical Analysis Results Scratch migration assay Proliferation assay Discussion Conclusion List of Abbreviations Competing Interests Authors Contributions Acknowledgements References Figures Tables 76 viii

9 CHAPTER IV: An In Vitro Method to Evaluate the Effect of Low-Level Laser Therapy (LLLT) on the Expression Level of MiRNA-21 Expression in a Canine Skin Model 4.1 Introduction Materials and methods Cell culture Study protocols Low-level laser therapy Micro-RNA isolation and microrna analysis Statistical Analysis Results Discussion List of Abbreviations Footnotes References Figues 84 CHAPTER V: Summary and Conclusion References 89 CHAPTER VI: Appendices 6.1 Figures Tables 96 ix

10 LIST OF TABLES Table 1 Table 2 Table 3 Low-level laser therapy protocols for all groups for the scratch migration assay and proliferation assay Scratch migration assay table of statistical analysis of the linear mean length of all groups over time Proliferation assay table of statistical analysis of the mean absorbance of all groups over time Page x

11 LIST OF FIGURES Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Effect of LLLT on wound size immediately following laser therapy. Linear mean length with the confidence interval for each wounded area is presented. Effect of LLLT on wound size 12 hours following laser therapy. Linear mean length with the confidence interval for each wounded area is presented. Effect of LLLT on wound size 24 hours following laser therapy. Linear mean length with the confidence interval for each wounded area is presented. Effect of LLLT on wound size 36 hours following laser therapy. Linear mean length with the confidence interval for each wounded area is presented. Effect of LLLT on cellular proliferation immediately following laser therapy. Mean absorbance with the confidence interval for each group is presented. Effect of LLLT on cellular proliferation 24 hours following laser therapy. Mean absorbance with the confidence interval for each group is presented. Effect of LLLT on cellular proliferation 48 hours following laser therapy. Mean absorbance with the confidence interval for each group is presented. Effect of LLLT on relative expression of mir-21 normalized to snrna U6 reference gene 24 hours following laser therapy. Mean relative expression with standard error of the mean for each group is presented. Photographs representative of the different cellular concentration of canine epidermal keratinocytes seeded in 6-well acrylic plates for the scratch migration and proliferation assays. Page xi

12 Figure 10 Figure 11 Photograph demonstrating a scratch created in a canine epidermal keratinocyte cell monolayer for the scratch migration assay, at the beginning of the experiment. Representative scratch migration assay of canine epidermal keratinocyte cells at the 12-hour time point Figure 12 Photograph of the high-power class IV Helium-Neon (He-Ne) laser 95 system unit (hand piece) maintained in a custom-made stand holder. xii

13 LIST OF ABBREVIATIONS ATP bfgf CPEK cdna EGF FBS FGF FDA GaAlAs Nd:YAG He-Cd He-Ne InGaAs LLLT mir/mirna mrna NO PBS PDGF qpcr qrt-pcr Adenosine triphosphate Basic fibroblast growth factor Canine epidermal keratinocyte progenitors Complementary deoxyribonucleic acid Epidermal growth factor Fetal bovine serum Fibroblast growth factor Food and drug administration Gallium-Aluminum-Arsenide Neodymium Yttrium Aluminum Garnet Helium-Cadmium Helium-Neon Indium Gallium Arsenide Phosphide Low-level laser therapy Micro-ribonucleic acid Messenger ribonucleic acid Nitric oxide Phosphate buffered saline Platelet-derived growth factor Quantitative real time polymerase chain reaction Quantitative reverse transcription polymerase chain reaction xiii

14 RCBD RNA ROS TGF-β VEGF WST-1 Randomized complete block design Ribonucleic acid Reactive oxygen species Transforming growth factor β Vascular endothelial growth factor Water-soluble tetrazolium xiv

15 CHAPTER I Literature review 1.1 Physiology of wound healing In mammals, the skin is the most voluminous organ of the body and is composed of two primary layers; the epidermis and the dermis [1]. The epidermis is the outermost or superficial layer, serving as the physical barrier between the interior body and exterior environment [1]. It is composed mainly of keratinocytes but dendritic cells, melanocytes, and Langerhans cells are also present [1]. The dermis, beneath the epidermis, provides the structural support of the skin and contains the connective tissue, hair follicles, sebaceous and sweat glands [1,2]. The principal function of the skin is protection of the interior body against the exterior milieu, maintaining a homeostatic internal environment [1-3]. Cutaneous wounds are the result of damaged or disrupted skin integrity and are most often caused by traumatic injuries, including surgical incisions and thermal injuries [4]. Wounds may be superficial also termed partial-thickness involving only the epithelium, or may be full-thickness, extending through the dermis and deeper into the subcutaneous tissue damaging other adjacent structures such as muscles, tendons, ligaments, bone, vessels and nerves [5]. Wound healing is the physiologic process of cutaneous tissues to restore structural integrity and function following any type of injury [5,6]. The healing process constitutes a dynamic tissue reaction, involving multiple and programmed biological stages including hemostasis, inflammation, proliferation, and 1

16 tissue remodeling [6-8]. Although wound healing progresses through different stages at different times following injury, it is likely that many phases are occurring simultaneously [8]. Wounds may be broadly classified as acute or chronic, but no universally accepted or acknowledged definition to differentiate them currently exists in the medical literature [8,9]. Acute wounds are generally tissue injuries that complete the wound repair process within the expected timeline, and through the predictable and programmed phases of wound healing [6,8,9]. Chronic wounds are tissue injuries that do not heal in an orderly and timely healing process; the healing repair is incomplete and may be disturbed by numerous factors [9,10]. Impaired-healing wounds have indeed failed to progress through the programmed physiologic phases of wound repair, impeding restoration of anatomical and structural skin integrity following traumatic injury [9,10]. Despite the fact that cutaneous lesions vary in nature, wounds have a common fundamental mechanism for repair and healing [5,10]. For each phase of the wound healing process, anatomical and functional properties of the injured skin are restored by the actions of numerous cellular components including platelets, neutrophils, monocytes/macrophages, endothelial cells, fibroblasts, myofibroblasts, and keratinocytes, and their interactions with various biochemical mediators such as growth factors and cytokines [11,12]. Growth factors and cytokines include a large and diverse family of polypeptide regulators, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor β (TGF-β), and vascular endothelial growth factor (VEGF) [11,12]. They are produced widely throughout the body by different type of cells and are 2

17 key for cellular communication, migration and proliferation during the wound healing process [12] Phases of wound healing i) Hemostasis Immediately following skin injury, a complex cellular and molecular response to the damaged vascular endothelium is established to prevent further exsanguination from the wound and microorganism invasion from the surrounding environment [1-5]. This process is called hemostasis and is the first phase of wound healing. It includes vasoconstriction, platelet aggregation and degranulation, and the formation of a thrombus also called a clot from the activation of the coagulation cascade [5,13,14]. The thrombus is the end product of the hemostatic process [14]. Hemostasis is initiated by a variety of factors and may be divided into primary hemostasis and secondary hemostasis [15]. Primary hemostasis involves the interaction between platelets and the damaged endothelium, resulting in the formation of a platelet thrombus [15]. Secondary hemostasis encompasses the activation of coagulating factors that lead to the formation of fibrin [15,16]. Fibrin s primary role is to solidify the platelet thrombus [15,16]. Endothelin and other vasoactive amines are then released by the disrupted vascular endothelium and induce the contraction of smooth muscle within peripheral vessels [15-17]. Vasoconstriction prevents hemorrhage and electrolyte loss from the wound site and aids platelet aggregation [15,17]. Platelets adhere to the exposed and damaged vascular subendothelial collagen and to each other through adhesive mediators such as fibrinogen, fibronectin, and von Willebrand s factor, resulting in 3

18 primary hemostasis by the formation of a platelet plug [15-17]. These aggregated and activated platelets then release the contents of their alpha granules, in a process called degranulation [5,15]. Platelets alpha granules contain epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and many other important cytokines, attracting additional platelets required for hemostasis to the local injured environment [13,17]. The coagulation cascade is composed of intrinsic and extrinsic components and is activated via various factors during the hemostatic process [16]. The intrinsic coagulation cascade is initiated by activation of plasma factor XII, which occurs when the damaged vascular endothelium is exposed to the extravascular milieu, whereas the extrinsic cascade is initiated by exposure of tissue factor expressed on extravascular cellular surfaces, activating plasma factor VII [16]. The extrinsic cascade is the main coagulation pathway in trauma-induced coagulation and wound healing; the intrinsic pathway is therefore not indispensible [15,16]. Both coagulation cascades may activate factor X, referred to as the common pathway, which together with cofactor Va, activate prothrombin to thrombin, converting plasma fibrinogen to fibrin [16]. Insufficient or inadequate fibrin formation causes impaired wound healing, as fibrin is the principal component of the mature thrombus [14]. Fibronectin, with its multiple binding sites for cellular attachment, is an adhesion protein and another important component of the mature thrombus [15,16]. Fibronectin in the presence of activated factor XIII will bind to fibrin, forming a provisional extracellular matrix (ECM), which guides and supports cellular migration to the affected area during the wound healing process [15,16]. 4

19 Thrombus formation is therefore an essential stage for any wound healing as it provides a scaffold for cellular migration, prevents further hemorrhage, prevents fluid and electrolyte loss from the wound site, and limits contamination from the extracorporeal environment, preventing infection [1,5,15]. Hemostasis is a critical phase of any wound healing process, as the repair cannot be completed until it is fully achieved [5,14]. ii) Inflammation The second phase of wound healing is inflammation [1-5]. The short, immediate vasoconstriction period is followed by a more persistent period of vasodilation and increased vascular permeability, creating the classic signs of inflammation: pain, heat, erythema and edema [5, 13-15]. Vasodilation results in a rapid migration of inflammatory cells and essential factors for wound healing to the injured site [5,15]. The transition from vasoconstriction to vasodilation is mediated by a variety of factors including leukotrienes, prostaglandins, and histamine [4,5]. Inflammatory cells, such as neutrophils and monocytes, are attracted to the wounded area by growth factors and cytokines, which are released mainly by activated platelets but also by damaged cells at the wound site [1,5,15]. This process of chemical cellular attraction is known as chemotaxis [18]. Neutrophils are the first inflammatory cells to arrive in the wound and are usually present within 24 to 48 hours of injury [5,15]. Neutrophils have many functions including elimination of bacteria through release of reactive oxygen species (ROS), phagocytosis of degraded bacteria and necrotic debris, as well as breakdown of the damaged extracellular matrix through the release of matrix metalloproteinases (MMPs), such as collagenase and elastase [1,5,15]. Neutrophils predominate for the first few days following the injury and 5

20 then disappear unless the wound becomes infected [13]. In the presence of infection, neutrophilic infiltration will continue, prolonging the inflammatory phase until the infection is controlled, therefore delaying wound healing [13,18]. Approximately 48 to 96 hours following injury, monocytes that have differentiated to tissue macrophages have become the primary inflammatory cells in the wound [5,13]. Most neutrophils, at this point, have been phagocytized by these cells or have undergone apoptosis [5]. Apoptosis, also commonly called programmed cell death, occurs by the activation of cellular endogenous calcium-dependent proteases [14]. The activation of these proteases, more specifically caspases, produces a cascade of intracellular proteolytic activity resulting in nuclear fragmentation, chromatin condensation, and chromosomal deoxyribonucleic acid (DNA) fragmentation [14,18]. However, the exact mechanisms that lead to inflammatory cell apoptosis during wound healing have yet to be determined [14,19]. Macrophages, unlike neutrophils, are essential to wound healing [5,15]. Although both have very similar functions in debridement of the wound, macrophages are essential because they release additional cytokines and growth factors, facilitating further migration at the injury site of other important cells involved in wound healing, such as fibroblasts and epithelial cells [13-15]. Nitric oxide (NO), mainly released by macrophages, is thought to regulate collagen formation, cellular proliferation and wound contraction in wound healing [13-15,18]. The exact role of this short-lived free radical molecule in wound healing has yet to be fully determined, however, inhibition of its release has been found to impair wound healing [14,20]. Eosinophils and basophils are other inflammatory cells active at the site of the wound [14]. These leukocytes are non-specific and their role in the inflammatory phase of wound healing remains to be fully defined [14,15]. Following inflammation and 6

21 wound debridement, wound healing progresses in a repair or proliferative phase [5,6,15]. iii) Proliferation The cellular milieu in the wound changes noticeably in the 7 days following injury [13,15]. The transition from the inflammatory phase to the proliferative phase is manifested by the invasion of fibroblasts, endothelial and epithelial cells, as well as an increased accumulation of collagen at the site of injury [15]. The main purpose of the proliferative phase is permanent closure of the wound and may last as long as 14 to 21 days [6,13,15]. Several processes occur during this phase, including angiogenesis, granulation tissue formation, epithelialization, and wound contraction [5,6,15]. Angiogenesis, also called neovascularization, is the growth and formation of new capillaries from the existing vasculature adjacent to the wound edges [4,13,15]. Angiogenesis occurs in response to tissue hypoxia and the production of growth factors, mainly vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bfgf), by cells within and around the wound [14,15]. Closure of the wound surface via formation of the mature thrombus during hemostasis is necessary to create this desired hypoxic wound environment [4,14]. Lactic acidosis and macrophages secreting angiogenic growth factors also simulate angiogenesis [13,14]. Capillary ingrowth results in the development of a microvascular network within the wound that provides oxygen and nutrients required by the migrating and proliferating cells during the repair process [13-15]. Simultaneously with angiogenesis, re-epithelialization of the damaged area occurs as a result of fibroblast migration from surrounding tissues and their proliferation in the 7

22 wound, in response to platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and epidermal growth factor (EGF) [6,13]. The scheme of processes involving fibroblast proliferation, migration in the wounded milieu, and production of proteins and collagen that contribute to the formation of granulation tissue is known as fibroplasia [15]. Undifferentiated cells within and at the periphery of the wound may also transform into fibroblasts under the effect of various cytokines and growth factors present at the injured site [4,6]. Fibroblasts are mainly responsible for the synthesis of collagen during the healing process, but they also participate in the contraction of the wound following their differentiation into myofibroblasts [4,13]. Collagen is the main structural protein constituent of wound connective tissue, making up 25% to 35% of the whole-body protein content [4,15]. Collagen plays a key role in wound healing, especially in the proliferative and remodeling phases of the repair process [4,5,15]. The activity of fibroblasts at the injured milieu causes the provisional ECM composed of fibrin and fibronectin to be gradually degraded and replaced with a more mature collagenous matrix and this transition is effective by 3 to 5 days after injury [4,14,18]. Fibroblasts that have infiltrated the wound site actively produce type III collagen, proteoglycans, glycoaminoglycans, and fibronectin, which are the principal components of the mature ECM [15,18]. The mature matrix promotes cellular adhesion and migration, in addition to tissue hydration [15]. Intermolecular bonding then ensues between the collagen fibers, resulting in an extracellular collagen matrix that is resistant to destruction [6]. The formation of new blood vessels establishing a capillary network, along with fibroblasts, inflammatory cells, and the different components of the ECM compose the 8

23 granulation tissue, which is the macroscopic feature of the proliferating phase [6,15]. The appearance of granulation tissue, macroscopically, differs from red and granular to white and nodular based on the vascular and collagen content of the injured milieu [15,18]. Wounds involving tissues with low blood supply, such as periosteum, fascia, tendons and ligaments produce granulation tissue slowly compared to highly vascularized wounds [15]. Under the influence of various cytokines and growth factors, epidermal cells, such as keratinocytes, migrate within the extracellular-collagen-matrix scaffolding or granulating tissue, and then proliferate for re-epithelialization of the wound [5,15,18]. Usually, new epithelium is not apparent at the periphery of the wound until 3 to 5 days following cutaneous injury [5,6,15]. Re-epithelialization of a wound is complete when epidermal cells have entirely covered the surface of the skin defect [15,18]. At that time, the wound is considered closed [15]. In partial-thickness skin wounds, keratinocyte migration over the cutaneous defect is initiated immediately following the injury from the wound edges and adnexal appendages, such as hair follicles, or sweat and sebaceous glands [14,15]. In full-thickness skin wounds, re-epithelialization may only occur following formation of adequate granulation tissue [14,15,18]. Migration occurs from the wound margins towards the center in a centripetal movement [6,15]. The migration and proliferation of the epidermal cells continue until contact is achieved with other cells migrating from different directions [4,6]. At the point of contact, cellular migration ceases and this process is known as contact inhibition [4,14]. Once migration is completed, epidermal cells form solid attachments to each other and the basement membrane and then resume the process of terminal differentiation for the formation of a stratified epidermis [5,6,14]. 9

24 The final component of the proliferative phase is wound contraction, resulting in a diminution of the wound size and changes in the tension of the wound and surrounding tissue [5,6,15]. The rate of contraction varies between anatomic locations and depth of the skin defect [15]. Wound contraction is evident by 7 to 9 days following cutaneous injury [14,15]. Fibroblasts that are anchored within the wound are transformed into myofibroblasts in response to numerous factors, mainly TGF-β [5,15]. Myofibroblasts, which are similar to smooth muscle cells, are required for wound contraction [14]. The main characteristics of myofibroblasts include a multi-lobulated nucleus, actin-rich microfilaments in the cytoplasm, and abundant rough endoplasmic reticulum [4,14]. Myofibroblasts contract by using smooth muscle type actin-myosin complexes, called alpha-smooth muscle actin, speeding wound repair by contracting the margins of the wound [14,15]. Contraction is a key stage of wound repair as it facilitates migration of epidermal cells by reducing their trajectory, thereby reducing the wound healing time [4]. Wound contraction in the proliferative phase ends when myofibroblast contraction is inhibited by contact of the wound edges and their disappearance is suspected to be via apoptosis [14,15]. Low myofibroblastic activity in the granulating tissue may result in wound contraction failure and delayed wound healing [14,15]. Excessive wound contraction is sometimes associated with a pathologic process known as wound contracture, limiting motion of the underlying tissues [6,14]. Wound contracture may be of particular concerns for wounds identified on the limb where it can limit joint mobility and function, as well as form a physical tourniquet, causing impairment of venous drainage, which leads to limb edema [6,14,18]. When the levels of collagen production and degradation are similar, the maturation/remodeling phase of tissue repair is initiated 10

25 [4-6,14,15]. During maturation, type III collagen, which is prevalent during proliferation, is gradually replaced by type I collagen [6,14,15]. iv) Remodeling or maturation The final phase of wound healing is remodeling or maturation of the granulating tissue [13-15]. Wound remodeling predominates as the primary wound healing activity approximately 19 to 21 days following skin damage and it may continues for months to years, depending on the type of cutaneous injury [6,14,18]. During this phase, type III collagen decreases from 30% to 10% and is slowly replaced by type I collagen [13]. The originally disorganized collagen fibers are rearranged, cross-linked, and aligned along the lines of tension [6,13]. In addition, the cellular density within the wound is reduced via apoptosis and the number of blood vessels in the wounded area regresses [13-15]. These changes result in the formation of a strong and acellular scar tissue [6,15]. As the phase progresses, the tensile strength of the wound increases, but the scar tissue never achieves the strength of the original, intact, uninjured skin and the adnexal structures do not usually regenerate [6,13-15]. At maximum tensile wound strength, scars are only about 70% to 80% as strong as uninjured tissues [6,13-15]. 1.2 History of light therapy The use of natural light as a healing modality, called phototherapy, was first reported many centuries ago [21]. While light therapy had no scientific origin at the time, natural sunlight was used by many civilizations for the treatment of various diseases [21]. Sir Isaac Newton was the first to show, in the 17 th century, that light is composed of 11

26 different coloured components by shining a beam of white light through a prism [21,22]. Based on this discovery, Newton introduced the term colour spectrum and successfully demonstrated that every colour has a single angle of refraction, and that the spectrum of white light contains six colours arranged in a specific order; red, orange, yellow, green, blue and violet [21-23]. In 1864, James Clerk Maxwell, acclaimed as the father of modern physics, demonstrated one of the most successful theories in the history of physical sciences; the theory of electromagnetism [23]. Electromagnetic radiation is composed of electromagnetic waves, which are described as self-propagating oscillating waves of magnetic and electric fields travelling at the speed of light in a vacuum [23,24]. Maxwell s famous four mathematical equations, simply known as Maxwell s Equations, established the basics of electricity and magnetism [21]. Light therapy as it is known today would never have been possible without the understanding that light is a form of electromagnetic radiation [24]. introduced the quantum theory [21]. In 1900, Max Karl Ernst Ludwig Planck Planck showed that the energy of light is proportional to its frequency, and that light exists in discrete quanta of energy [21,22]. The Nobel Prize in Physics 1918 was awarded to Max Planck "in recognition of the services he rendered to the advancement of Physics by his discovery of energy quanta" [26]. In 1905, Albert Einstein proposed the existence of the photon, an elementary particle denoting a quantum of light [22,23]. In 1917, Einstein published the idea of stimulated light emission that eventually inspired the development of the laser [22-24]. The term laser originated as an acronym for Light Amplification by Stimulated Emission of Radiation, and was named by Gordon Gould [26,27]. The biological effects of light therapy, however, were not discovered until over 40 years after Einstein s discovery of 12

27 stimulated light emission [23,27]. It is only in 1960 that the physicist Theodore Harold Maiman created the world s first operative laser from excitation of a ruby crystal by a helical high-power flash lamp, from which the laser energy emerged [23]. Maiman is the author of The Laser Odyssey, which describes in detail the creation of the Ruby laser, first operated at the Hughes Research Laboratories in California [23]. In 1967, Hungarian physician and professor Endre Mester was the first to demonstrate the biological effects of low-level laser therapy (LLLT) by applying light to the backs of shaven mice [23, 27]. Mester noticed that the shaved hair grew back more quickly on the treated group than the untreated group, with no adverse side effects noted [23]. He then focused his work to the effects of laser therapy on wound healing in mice [23,27]. In 1971, Mester applied his successful animal experimental findings to treating human patients with non-healing skin ulcers, in which he was again successful [22,23,27]. From then on, Mester dedicated himself to studying the phenomenon of LLLT and was the first to present the photobiostimulation effect, which is the application of light within the red and infrared spectrum over injuries and lesions to stimulate healing and promoting pain relief within those tissues [23,27]. 1.3 Laser basics A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation [28]. A laser is constructed from three essential components: an energy source, a lasing/amplifying medium, and an optical resonating cavity bounded by mirrors [28, 29]. Lasers have certain unique properties compared to the light from other sources, allowing for penetration of light at the cellular 13

28 or molecular level [27-29]. Compared to other electromagnetic radiations, the light of a laser is monochromatic, coherent and collimated [28-30] Monochromaticity Being monochromatic, laser light is composed of light of only one wavelength compared to the light from other ordinary sources of which it is composed of many different wavelengths [29-31]. Indeed, the light of a laser has a single spectral colour and is the purest monochromatic light obtainable [27,29] Coherence The theory of coherence in physics may be explained by the concept of spontaneous and stimulated light emission [30-31]. When an atom transitions spontaneously to a lower energy level, a photon of random phase and direction is emitted [24,26]. This type of energy emission occurs naturally and in multiple directions at a time and is called spontaneous emission [24-26]. Photons emitted from conventional light sources are created by atoms without any associated phase and direction between each other and are therefore not coherent [24,28-31]. In stimulated emission, a photon may stimulate an atom to transition again, from higher energy level to lower level, but resulting this time in the emission of another photon of the same energy [26-28]. Indeed, a photon emitted by stimulated emission has the same phase, frequency, and direction as the original photon [26,30]. This identical phase relationship amongst travelling photons results in the laser beam generated having the property of coherence [31]. 14

29 1.3.3 Collimation The light from laser sources is also collimated because it is formed in an optical resonance cavity between two parallel mirrors, in addition to being monochromatic and coherent [28-30]. A collimated light describes a light beam that does not disperse with distance, or does so only minimally [24,28]. Collimation therefore refers to the propagation of light for which the beam remains parallel with distance [24,28-30]. 1.4 Laser physics The numerous types of laser units that have been developed so far present a wide range of physical and operating parameters [31]. A detailed description of laser physics is beyond the scope of this thesis but it is important to briefly describe some laser parameters; wavelength, energy, power, spot size, power density, and pulsed mode. All of these functions, in addition to timing of the applied light, must be selected for each treatment application, and basic knowledge of these terms are required Wavelength The concept of wavelength is important, as the biological cellular effect of laser therapy seems to be closely related to the wavelength of the light emitted by the laser unit [32]. In physics, a wavelength is the distance between one peak or crest of a wave of light, heat, or other energy, and the next corresponding peak or crest [28,32]. The wavelength is more often measured in nanometers, and is often designated by the Greek letter lambda (λ) [30,32]. 15

30 1.4.2 Energy In the context of physical sciences, several forms of energy have been described. These include thermal energy, chemical energy, electric energy, radiant energy, nuclear energy, kinetic energy, and magnetic energy [24]. The joule (J) is the International System of Units (SI) for energy [27, 31]. Energy is subject to the law of conservation of energy, which means energy can neither be created nor destroyed; it can only be transformed [30]. The total energy of a system remains constant over time [27,30] Power The power is expressed in Watts (W) [29,30]. It is the rate at which the energy is emitted from a laser unit, not the quantity of energy itself [33]. Power = Energy (Joule)/Time (s) Spot size The spot size is defined as the surface area of the collimated laser beam [33]. It is measured in cm 2 [32,33]. The spot size = πr (cm) 2, where π = 3.14 and r = radius (cm) Power density Power density, also called irradiance, is the amount of power (time rate of energy transfer) per unit surface area [33]. Power density is usually expressed in terms of W/cm 2 [30,34]. The irradiance of a laser unit system is much greater than a conventional light 16

31 source of the same power, as the light is coherent and collimated [34]. Power density = Power (Watts) / Spot size (cm 2 ) Continuous and pulsed emission Continuous emission is an electromagnetic wave of constant amplitude and frequency [30,31,34]. Pulsed emission refers to light produced from a laser source that appears in pulses of some duration and repetition rate [31,35]. Pulsed mode compared to continuous emission allows a short pause period between laser pulses [35]. Many types of laser units may operate in either wave mode [31,35]. There is no consensus on which light emission is best, and the choice remains largely based on personal preference [35] Laser classifications The most often employed lasers that promote wound healing are gas lasers, including helium-cadmium (He-Cd) and helium-neon (He-Ne), as well as diode lasers such as arsenium-aluminum (AsGa) and aluminum gallium arsenide (GaAlAs) [28, 36]. It is acknowledged, however, that the success of LLLT is dependent on time of application, wavelength, power, and dosage, not on the type of the laser unit system itself [37]. 1.5 Low-Level Laser therapy The treatment of injured tissues still remains quite a challenge in both veterinary and human medicine [38]. The care and management of acute and most importantly chronic cutaneous wounds are time consuming and may have significant health and 17

32 economic consequences [2,38]. Ongoing research has advocated new therapy options in order to accelerate and promote the tissue repair process. Of these, extracorporeal shockwave therapy, ultrasound therapy, and LLLT have been examined as means to accelerate healing of damaged tissues and improve clinical outcomes. Low-level laser therapy has been used in human medicine for almost fifty years, and is becoming increasingly popular in veterinary practices throughout the world [32,36]. This treatment modality may be described in the literature by any as of the following synonyms [35,36]: o Cold laser therapy o Soft laser therapy o Low-power laser therapy o Therapeutic laser therapy o Biostimulation laser therapy o Low-intensity laser therapy o Low-reactive laser therapy o Phototherapy o Light therapy o Low-energy photon therapy o Medical laser therapy Low-level laser therapy uses electromagnetic radiation within the visible red or near-infrared light to alter cellular function and enhance healing of injured tissues and improve clinical outcomes [36-38]. Low-level laser therapy applies low-power monochromatic, coherent and collimated light to various type of lesions [32,36]. It causes low or imperceptible temperature changes as opposed to high-energy lasers such as those used in surgery, which elicit their effect through laser-generated heat [39]. When used on injured tissues the energy absorbed by the cellular components of the area is not 18

33 transformed into heat or vibration, but into enhancement of intracellular photochemical and photobiological processes [39,40]. This process associated with LLLT is referred to as biostimulation or photobiomodulation [39,40]. At first used predominantly for wound healing, pain relief and reduction of inflammation, the medical applications of LLLT have expanded to include the treatment of many diseases such as neurological processes, musculoskeletal injuries, soft tissue injuries, abscesses, dental diseases, and various dermatologic ailments [32,39,40]. In recent years, it has become an increasingly used treatment modality, mainly in physical medicine and rehabilitation therapy [41]. The use of light therapy on tissue regeneration continues to gain in popularity, but there is no collectively accepted theory that defends all its cellular effects and biological processes in wound healing [40-42]. Numerous publications in the literature have reported beneficial results of LLLT in wound healing, although there are also some studies that showed no effect [42,43]. No study has ever fully explained the underlying etiology of light therapy [44] Suggested cellular and tissular mechanisms of laser therapy At the cellular level, the effect of LLLT is believed to be absorption of red and near-infrared light by the cytochrome c oxidase located in mitochondria, although the exact mechanism of action remains unknown [40,42,44]. In biology, the mitochondrion is defined as the cellular organelle that produces the energy required for cellular metabolism [45,46]. The respiratory electron transport chain of mitochondria is the main focus of this complex process and contains four complexes of integral membrane proteins; NADH dehydrogenase (Complex I), succinate dehydrogenase (Complex II), cytochrome c 19

34 reductase (Complex III), and cytochrome c oxidase (Complex IV) [44,46,47]. The production of energy adenosine triphosphate (ATP) in mitochondria, is conducted by processing oxygen and organic compounds through the electron transport chain [44,46]. The enzyme cytochrome c oxidase or Complex IV is the last enzyme of the mitochondrial electron respiratory chain [47,48]. Absorption of visible and near-infrared light is believed to result in an improvement of cellular metabolism via activation of the respiratory chain [39-42,44,48]. It is thought that the absorption of energy by the cytochrome c oxidase may improve electron transport, production of ATP, modulation of ROS, induction of transcription factors, and release of NO [44,48]. This cascade of metabolic effects results in many physiological changes, which in turn is thought to improve tissue repair and reduce inflammation and pain [40,42,44]. Only a few experiments that have focused exclusively on the mechanism of LLLT have shown that cellular respiration is upregulated when mitochondria are exposed to light in the red and near-infrared spectral range [48,49]. Chen and colleagues (2008) demonstrated in their study on endothelial cells that low-energy laser irradiation increases endothelial cell proliferation, migration, and endothelial nitric oxidase synthetase (enos) protein expression through activation of the PI3K/Akt pathway. At the cellular level, laser therapy improved cellular metabolism [49]. Silveira et al. (2006) successfully demonstrated, by mitochondrial enzyme evaluation, that red and near-infrared light delivered for 10 days to iatrogenic wounds created on the back of adult male Wister rats significantly increased the activity of the respiratory chain enzyme complexes II and IV (cytochrome c oxidase) [48]. The results of these analyses are in accordance with other data from literature where the cytochrome c oxidase is thought to be a key photoreceptor 20

35 of light in the red to near-infrared spectrum; this complex component of the mitochondrial respiratory chain seems to be activated by laser therapy [39-42,44,48,49]. However, more studies are required to fully evaluate mitochondrial enzymatic activities following light therapy in order to make any definitive conclusions. Low-level laser therapy remains controversial due to an incomplete understanding of the underlying etiology responsible for the observed beneficial effects [40-42]. Additionally, there are a large number of different irradiation protocols found in the literature, which makes the comparison of results among studies challenging [36,44]. For these reasons, light therapy remains an alternative treatment and its use is largely empirical [36,50] Biphasic dose-response of light therapy There are numerous publications in the literature that have shown beneficial results associated with LLLT in wound healing [36,50]. However, studies with less convincing results have also been published [50-52]. Treatment failure in certain circumstances can be attributed to several factors including inappropriate laser dosimetery, location of the wound, physiologic state of the patient, and concurrent medical therapy [51,52]. A large number of irradiation parameters such as the wavelength (nm), power (W), power density (W/cm 2 ), energy density (J/cm 2 ), emission (continuous or pulsed), and illumination time (s) of the applied light must be selected for each treatment [42,50,51]. It is well recognized that LLLT using appropriate protocols improve wound healing; a suboptimal selection of laser parameters may result in a negative or less favorable therapeutic outcome [50-53]. There is therefore an optimal 21

36 balance of energy density and time that causes a maximal beneficial effect; a doseresponse relationship to light therapy has been previously recognized [50-54]. A biphasic dose-response has been observed both in animal and culture cell models, where low levels of light have a better effect on stimulating healing than higher levels [42,51,52]. The Arndt-Schulz curve is a three dimensional graphical illustration frequently cited as an appropriate model to illustrate this biphasic dose-response seen with laser therapy [51,52]. The biphasic dose-response is function of energy density also called irradiance and light time [51,52]. Some clinical experiments demonstrated the phenomenon of biphasic doseresponse, and this is one of the main reasons why there is disagreement between clinical results and lack of clear conclusions regarding the observed effects of laser irradiation therapy [51-56]. Hawkins and Abrahamse (2006) used HeNe laser (632.8 nm) with irradiation doses of 0.5, 2.5, 5, 10, and 16 J/cm 2 in their work to establish the behavior in vitro of human skin fibroblasts. They demonstrated that a single dose of 5 J/cm 2 caused increased mitochondrial activity, cellular proliferation and viability, without adversely causing cellular damage; higher doses (10 and 16 J/cm 2 ) resulted in an inhibitory effect with a significant amount of cellular damage identified [54]. Houreld and Abrahamse (2008) used HeNe (632.8nm), GaAlAs (830nm), and Nd:YAG (1064nm) lasers for healing of in vitro wounded diabetic-induced human skin fibroblasts irradiated with either 5 or 16 J/cm 2. Regardless of the wavelength used, all cells irradiated with 16 J/cm 2 showed incomplete wound closure, increased apoptosis, and decreased basic fibroblast growth factor expression. The fibroblasts responded better when irradiated with an energy density of 5 J/cm 2 at a wavelength of nm [55]. Basso et al. (2012) 22

37 demonstrated that irradiation with a InGaAsP diode laser (780nm) in cultured human gingival fibroblasts with energy doses of 0.5 and 3 J/cm 2 resulted in a significant increase in cellular metabolism compared with the non-irradiated control group and irradiation with higher energy doses (5 and 7 J/cm 2 ) [56]. Animal model experiments have also shown similar dose-dependent responses, leading to discrepancies in clinical outcomes observed based on the irradiation protocol selected [42,51,52]. Demidova-Rice and collaborators (2007) documented this phenomenon when they tested the effect of 635 nm non-coherent light at 1, 2, 10 and 50 J/cm 2 on full-thickness dorsal excisional wound in mice. Comparison of the area under the healing curves generated based on the wound healing rates over time among groups revealed that a single exposure with 1, 2 and 10 J/cm 2 improved wound healing compared to the non-irradiated control group, while the group irradiated with 50 J/cm 2 presented an inhibitory effect [42]. The concept of biphasic dose-response is important in LLLT and the complexity of choosing amongst a large number of illumination parameters may explain the publication of a number of negative studies [42,51,52,56]. Low-level laser therapy can modify cellular processes to have a stimulatory or inhibitory effect [55,57]. In other words, LLLT has a dose-dependent capability to alter cellular behavior. There are no accepted standard protocols for the use of laser therapy in the literature at this time [50,51] The biological effects of laser therapy Wound healing was one of the first applications of laser therapy following its 23

38 discovery [58]. It has been shown that the use of this treatment modality on cutaneous wounds presents several advantages, such as the control of pain, reduction of the inflammatory response, modulation of the immune system, and acceleration of wound healing [36,59]. Mester and colleagues, while working at Semmelweis University in Budapest, Hungary, were the first to use laser therapy to treat patients with non-healing skin ulcers [58,60]. They were the first to report significant improvement in wound healing using various laser systems and they are the pioneers of LLLT in human medicine [58,61]. Although their early reports lacked appropriate control groups for comparison, high-quality design studies in humans followed; some with positive results and others with no significant difference [41,62]. Kajagar and colleagues (2012) conducted a randomized, controlled trial to evaluate the efficacy of LLLT on wound healing in sixtyeight patients with chronic diabetic foot ulcers. Based on the measured percentage wound area, they saw that all ulcers treated with laser irradiation contracted significantly more compared to those in the control group [62]. Hopkins et al. (2004) demonstrated in their triple-blinded, controlled, experimental human-wound model study that low-level laser irradiation enhanced healing of partial-thickness lesions, as measured by greater wound contraction, in 22 treated patients [41]. A randomized, controlled study by Lundeberg and Malm (1991) evaluated the effect of HeNe laser delivered at a dose of 4 J/cm 2 in the treatment of chronic venous leg ulcers. The results of their experiment showed no significant differences in the proportion of healed ulcers or ulcer area in the HeNe group compared with the placebo group at the end of the study period [63]. Similarly, no significant variation in wound closure rate was observed by Lagan and colleagues (2001) in patients treated with GaAlAs (820nm) at 9 J/cm 2 compared to the group not treated by 24

39 laser therapy, in uncomplicated postoperative wounds following minor podiatric surgery [64]. i) Tissue repair One of the most frequently claimed beneficial effects of phototherapy is increased tissue repair and this effect is likely the main reason why laser therapy is now commonly used in many practices [32]. Low-level laser therapy may impact each of the different wound healing phases of inflammation, proliferation, remodeling and maturation, in a positive manner [44]. Low-level laser therapy is believed to enhance wound healing by increasing macrophage activity, neovascularization/vasculogenesis, cellular adhesions, fibroblast proliferation, and healed wound tensile strength [32,44,61]. Macrophages are essential to wound healing [13,15]. The primary role of macrophages is to phagocytize bacteria and debris from the wound environment by releasing proteases, and to secrete cytokines and growth factors [6, 13,15]. Low-level laser therapy increases macrophage activity and as a result, shortens the end of the inflammatory phase, allowing the proliferation stage of the wound healing process to begin early [13,65]. In a recent study published by Souza and collaborators (2014), the effect of LLLT on the mitochondrial activity of macrophages was evaluated. Macrophages were activated with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) for 24 hours to simulate an inflammatory process, and then irradiated with laser therapy using two sets of parameters (780 nm; 70 mw; 3 J/cm 2 and 660 nm; 15 mw; 7.5 J/cm 2 ). Non-activated and non-irradiated macrophages were used as a control group. Macrophage activity was determined using the cell mitochondrial activity (MTT) assay in 25

40 three predetermined, independent experiments (1, 3 and 5 days), and compared among each group. The results of their work showed that both laser treatment protocols could modify cellular activation status of macrophages [65]. Because of the relative ease of working with small animals, most studies using LLLT for wound healing have been conducted on rodent models [61]. Most experiments have, again with mixed results, focused on surgical and burn wound healing, skin-flap survival, and wound tensile strength [40,61]. Several lines of evidence have demonstrated increased rate and quality of wound healing following LLLT [36,41,53,62]. For example, Colombo and colleagues (2013) examined the healing effects of HeNe laser with a wavelength of 660nm delivered at dose of 10 J/cm 2 on open surgical wounds produced on the dorsum of twenty-four young adult male Wister rats. Animals were randomly distributed into control or treatment groups. Each group was further divided into three subgroups based on the predetermined animal s death time on the 2 nd, 4 th, or 6 th day following wounding. Laser therapy started following surgery and was repeated every other day until the animals were sacrificed. Histological analysis by light microscopy revealed that collagen expression and number of blood vessels were higher on irradiated animals than the control ones, indicating that laser therapy positively influences angiogenesis and collagen deposition in wound healing [53]. These results are supported by a similar animal model study performed by Tacon and collaborators (2011), where the wound healing activity of InGaAlP laser (660nm) in rats was evidenced by increased angiogenesis, decreased polymorphonuclear infiltrate and hemorrhage, as well as pronounced fibroplasia compared to the control group that received no irradiation [66]. Busnardo et al. (2010) demonstrated in their work on sixty Wistar rats with sutured, 26

41 longitudinal, and dorsal incision that the same inflammatory pattern was present among their non-irradiated control group and irradiated experimental group. The experimental group had, however, fewer inflammatory cells at all three evaluation times (3, 7 and 14 days) with faster reduction in their number. They also had greater and earlier total collagen density, with more type III collagen. The authors concluded that LLLT does not modify the quality of the inflammatory response, but shortens its duration, in addition to increases collagen synthesis in the early phases of the healing process [67]. Tensile strengh, another important aspect of wound healing, has been closely examined with the use of LLLT [61]. Low-level laser therapy may intensify the final tensile strength of the healed tissue by increasing the amount of collagen synthesis and by increasing cellular migration and proliferation [61,68,69]. Lyons and colleagues (1987) demonstrated substantial improvement in the wound tensile strength of irradiated wounded hairless mice using HeNe laser with an energy density of 1.22 J/cm 2 [68]. A more recent study by Vasilenko and colleagues (2010) also successfully demonstrated the positive effect of low-light therapy on wound tensile strength in forty young male Sprague-Dawley rats [69]. Low-level laser therapy not only decreased wound healing time, but also improved healed wound tensile strength [68,69]. In addition, wounds treated with this modality seem to heal with less scarring [70]. In dermatology, LLLT has beneficial effects on wrinkles, acne and hypertrophic scars, herpes virus infection, pigmentary disorders (vitiligo), and inflammatory diseases (psoriasis) [50]. In 2015, Kurach presented at the Michigan Veterinary Conference an abstract describing the effect of laser therapy compared to standard-of-care wound management on the healing of acute, full-thickness, open wounds created on the back of ten adult male 27

42 Beagles. This prospective, randomized and controlled experimental study is the first known project to evaluate the in vivo effect of LLLT in veterinary medicine. The author reported no significant difference between the two groups evaluated for all parameters, including total wound area, percent contraction, percent epithelialization, histologic acute inflammation scores, and histologic repair scores. In this study, there was no beneficial effect observed with the use of LLLT on acute wound healing in dogs. The laser parameters selected was however not mentioned in the abstract. ii) Pain relief The analgesic effects of red and near-infrared laser radiation are well documented in several laboratory experiments and human clinical studies [32,41,62,71]. Pain relief is provided through many different local and systemic pathways [32]. The effect of laser therapy in the relief of pain seems to be attributed to endogenous opioid (beta-endorphins) and NO release, and inhibition of the release of noxious mediators such as bradykinin [32,40,72]. Nitric oxide is an important inter- and intracellular messenger and a powerful vasodilator [20,73]. Vasodilation within a wounded area results in reduction of ischemia, improved cellular perfusion and enhancement of lymphatic drainage [73]. Nitric oxide associated vasodilation is thought to be the main cause of the transient analgesic effects caused by light therapy, but has yet to be entirely proven [14,44,49,73]. Endorphins are endogenous opioids that attach to µ-opioid receptor, obliterating all sensation of pain [72]. Increased peripheral beta-endorphins secondary to laser photobiostimulation has also been targeted as a possible mechanism of pain relief [72]. Hagiwara and colleagues (2008) 28

43 investigated, on their rat model of inflammation, the effect of laser therapy on peripheral endogenous opioid analgesia following laser photobiostimulation of blood. The expression of the beta-endorphin precursors was evaluated by quantitative reverse transcription polymerase chain reaction (qrt-pcr). Peripheral endogenous opioid production increased following laser therapy [72]. Additionally, pain modulation may occur due to alterations in nerve conduction by blocking depolarization of C-fiber afferent nerves [74]. Most experiments evaluating the analgesic effect of laser phototherapy were performed by looking at nerve conduction and regeneration in patients with ischemic stroke, neuropathic pain, or tendinopathy [35]. Over 30 experiments successfully demonstrated an analgesic effect of laser therapy in the literature. Within those, Ponnudurai et al. (1987) compared the analgesic effect, by rat tail-flick test, of He-Ne laser using various pulse repetition rates (4, 60, and 200 Hz). They noted a 50% increase in pain threshold following laser therapy. Light photobiostimulation with 4 Hz produced the most rapid and transient analgesia while the response to 60 Hz was delayed but persisted longer. Treatment with the highest pulse repetition rates of 200 Hz did not produce any hypoanalgesia [75]. Similarly, Sushko and colleagues (2007) reported analgesic property of laser phototherapy on the treatment of acute pain in mice, inflicted by hypodermic injection of formalin solution [74]. Low-level laser therapy is now widely used in physical therapy, chiropractic, and sports medicine for the treatment of various diseases, as it seems overall to significantly attenuate pain [40, 74,75]. 29

44 iii) Inflammation As previously mentioned, LLLT shortens the inflammatory phase of wound healing indicating that light may have anti-inflammatory properties [67,77]. Low-level laser therapy is thought to mimic the effects of anti-inflammatory drugs by various pathways, but mainly by decreasing levels of prostaglandin-2 (PGE 2 ) and inhibiting cyclooxygenase-2 (COX-2) [71,76,77]. A recent study conducted by Lim and collaborators (2015) demonstrated that both direct and indirect exposure of cultured human gingival fibroblasts with 635 nm light could inhibit activation of pro-inflammatory mediators, as COX-2 protein expression and PGE 2 production were significantly decreased compared to the non-irradiated control group [71]. Increased energy production, acceleration of leukocyte activity and NO-mediated vasodilation, allowing a more rapid removal of non-viable cellular and tissue debris, thereby increasing repair, also play important roles in reduction of the inflammatory response [44,49]. By decreasing inflammation, laser therapy appears to provide pain relief and facilitates the wound healing process [40,50,51]. 1.6 Laser safety and special considerations Low-level laser irradiation is a safe method of treatment in wound healing; there are no absolute contraindications with the exception of ocular exposure [34,40]. Eye protection must be worn by the therapist, any other supporting operators and the patient, at all times during the therapy session. Optical exposure and subsequent ocular damage, such as blindness, to laser light can be direct or indirect, such as by reflection of the rays on metallic surfaces [34,40]. All other specific indications for operating a laser unit are 30

45 based on responsibility and diligent application, not scientific data [40]. The Food and Drug Administration (FDA) approved low-level laser irradiation as a safe treatment modality [34]. It has no known side effects [34,40]. To avoid accidental and unnecessary exposure, a visible posted warning sign should always be present outside of the operating room. Hawkins and colleagues (2005) suggested that light therapy should ideally be avoided in patients with pacemakers, metastatic diseases or central neurological conditions, and they recommended to avoid irradiation over the thyroid gland, ovaries and testicles [40]. These authors have however not listed the specific reasons for its avoidance in these cases. 1.7 MicroRNA expression MicroRNA biogenesis and function In 1993, Victor Ambros and his two colleagues, Rosalind Lee and Rhonda Feinbaum, discovered the first MicroRNA (mirna or MiR) from a gene called lin-4, known to regulate larval development of Caenorhabditis elegans, a nematode [78]. A novel way of gene regulation was revealed. MicroRNAs are non-protein coding, single stranded ribonucleic acid (RNA) molecules composed of approximately nucleotides in length, so they are relatively short [79]. Their main function is to control posttranscriptional gene expression by binding to the 3 -untranslated region (3 -UTR) of specific and targeted messenger RNAs (mrnas), inhibiting their translation or inducing their degradation [79]. Although little is still known about mirnas, they seem to modulate more than one third of all protein-encoding mrnas of the human genome [80]. 31

46 The importance of mirna-gene regulation is gaining more attention and is now becoming the main focus of multiple on-going research projects because their function and significance are progressively discovered [79,80]. MicroRNA biogenesis usually occurs through specific and consecutive processes. [80-84]. This multi-step mechanism is first initiated with the RNA polymerase II enzyme, which is responsible for the production of several nucleotide-long fragments called the primary mirnas (pri-mirnas) [81,82]. This first phase of the biogenesis is called transcription [83]. The pri-mirnas produced are capped with a specific nucleotide at the 5 end, polyadenylated by addition of multiple adenosine nucleoside molecules and spliced [81,82]. The second phase is called export [83]. A nuclear complex comprising the RNAse polymerase III enzyme Drosha and DGCR8 have for role the cleavage of the pri-mirnas into short segments of premature mirna (pre-mirna) [81,82]. They are approximately 70 to 90 nucleotides-long [81-83]. The pre-mirnas are then actively exported from the nucleus through the cytoplasm by specific nuclear-transport mechanisms; the exportin-5 receptor and protein RanGTP [81,82]. At this location, the cytoplasmic RNAse III enzyme Dicer processes the pre-mirnas, resulting in the production of multiple double-stranded RNA molecules [81,82]. The formation of duplex RNAs is termed dicing [83]. In the last phase of mirna biogenesis, known as strand selection, interaction with the mirna-induced silencing complex (RISC) occurs, leading to the destruction of one of the strands of the RNA molecule; the other remains present and becomes the mature mirna [81-83]. The function of the mature mirna is to bind to target mrnas via complementary binding with a specific sequence in the 3 -UTR region, also known as seed sequence [81,82]. A single mirna may control many 32

47 different mrnas [81-84] MicroRNA in skin morphogenesis The skin is the largest organ of all and has as its principal function the protection of the interior body from the exterior environment [1,2]. It is composed of the epidermis, dermis and hypodermis [1]. MicroRNAs play a key role in many cellular processes and genetic regulation, and are extremely important in skin morphogenesis as well as dermal wound healing, through the regulation of cellular migration, development, proliferation, and apoptosis [81,82]. Yi et al. (2006) demonstrated in their work on embryonic skin progenitors that differential expression and regulation of numerous mirnas in the skin is necessary for normal morphogenesis and homeostasis [85]. For example, mir-200 and mir-205 have both been reported to be highly expressed in intact skin and have been shown to regulate the transcriptional repressors of E-cadherin, a tight junction protein that is essential for maintenance of the epithelial architecture of the skin [81,82]. MicroRNA levels may be increased or decreased, depending of the nature of the stimulus, and their expression or repression have been observed in multiple dermal diseases, neoplastic processes, and wound healing [86,87] MicroRNA in wound healing Many mirnas have been identified in the skin and generally appear to exert important functions many of which are not yet completely elucidated [81,82]. Among these, mir-205, mir-21, mir-122, mir-155, mir-200 family (mir-200a, mir-200b, mir-200c), mir-203a, and mir-205 are the most commonly reported [88,89]. 33

48 As previously described, the healing of damaged skin is a multi-step biological process involving three sequential phases; the inflammatory phase, the proliferation phase, and the remodeling phase [6-8]. The exact function and roles of mirnas in wound healing is not clearly defined at this time as limited numbers of reports are in the literature [81,82,90]. Ongoing work by Shilo et al. (2007) has suggested that the wound healing process is finely orchestrated by regulated changes in the expression of specific mirnas for each phase of the wound healing process [90]. For example, mir-147 has been shown to play a key role in avoiding excessive inflammation during the repair process and is therefore important during the inflammatory phase [91]. In a similar study, Lai and Siu (2014) reported that monocyte to macrophage differentiation was negatively affected by mir-424, decreasing the number of these phagocytic cells available for wound healing. Macrophages are necessary for the repair process to progress to the next phase [92]. It is well accepted that keratinocyte migration and proliferation are both essential for re-epithelialization to occur in the proliferation phase. MicroRNA-203, mir-205, and mir-21 were shown to accelerate cutaneous keratinocyte migration and proliferation in vitro through specific molecule targeting [93]. Wang et al. (2012) successfully demonstrated in adult C57BL mice, using mirna microarray analysis, that mir-21 expression was up regulated in wound healing. They also noted that its inhibition caused significant delay of wound closure secondary to diminished wound contraction and collagen deposition, and they therefore concluded that wound-induced mir-21 is crucial for the wound healing process to occur uneventfully [94]. MicroRNA-203 is known as the most prevalent keratinocyte-specific mirna in the epidermis by targeting the 3 -UTR of the transcription factor p63 [80]. Viticchiè and co-workers (2012) evaluated its 34

49 expression following creation of a wound in the epidermis of mice and they successfully showed that mir-203 has a specific role in wound healing by indirectly, but positively affecting keratinocyte migration and proliferation [80]. Van Solingen and collaborators (2014) evaluated the expression of mir-155 in wounded tissue compared to healthy skin in mice, 10 days following wounding. They noted that mir-155 expression, induced by the presence of inflammatory mediators such as macrophages, was up-regulated in their wounded groups compared to their healthy tissue groups, and that inhibition of mir-55 was associated with improved wound healing [95]. The specific mirnas involved in cutaneous wound healing have not been comprehensively identified at this time and their biological mechanism and function remain unclear [80, 88]. Further investigation of these mechanisms could eventually lead to the development of mirna-based therapies for the treatment of many conditions, including wound healing. Regulation of mirna-21 and mirna-203 gene expression has been proposed as a new therapy avenue in the treatment of non-healing and chronic wounds [88] MicroRNA and laser therapy Low-level laser therapy has been shown to have an effect on mirna expression, although very few published studies support this finding [96,97]. One of them performed by Wang et al. (2012) evaluated the effect of LLLT in an animal skin model by quantitative real-time PCR (qpcr), which revealed an increased proliferation of mesenchymal stem cells (MSCs), and a significant up and down regulation of many mirnas. The differential expression of these recognized mirnas changed over time as 35

50 analyzed by quantitative reverse transcription polymerase chain reaction (qrt-pcr) in a time-dependent mode. The expression level peaked between the second and fourth days following LLLT and then returned to normal concentration by eight days post-therapy. These authors also reported mir-193 has the most significantly up-regulated mirna following laser therapy [96]. Similarly, Kushibiki and his colleagues (2013) also evaluated the effect of light therapy in the induction of several mirnas gene expression in wound healing [97]. Overall, LLLT has been shown to induce mirna expression through a variety of cellular processes but further investigation is needed in this field to determine the mechanisms by which this induction occurs [96,97]. A detailed understanding of the specific mirna expression and regulation by laser therapy would represent an additional step towards our understanding of the exact mechanism of action of LLLT in wound healing [96,97]. 1.8 Rationale, Hypothesis and Objectives Low-level laser therapy is a clinical treatment that has received considerable attention and has gained popularity. It is mainly used in rehabilitation medicine and physiotherapy in human and veterinary medicine and uses low-levels of light in an attempt to improve the rate of tissue healing and clinical outcomes. The effect and specific mechanism of action of this treatment modality in wound healing is currently an active area of investigation. Also, much attention has been directed towards specific MicroRNAs involved in wound healing. 36

51 The authors hypothesized that laser therapy alters cellular activity through changes in proliferation, migration and regulation of mir-21 expression in an in vitro canine skin model. The objectives of this project were to: 1) Determine the effect of various LLLT doses on the migration of cultured canine epidermal keratinocytes in the healing of an in vitro canine skin model. 2) Determine the effect of various LLLT doses on the proliferation rate of cultured canine epidermal keratinocytes in vitro. 3) Determine the effect of various LLLT doses on the expression of MiR-21 of cultured canine epidermal keratinocytes in vitro. 1.8 References 1. Bielefeld KA, Amini-Nik S, Alman BA. Cutaneous wound healing: recruiting developmental pathways for regeneration. Cell Mol Life Sci. 2013;70(12): Martin P. Wound healing-aiming for perfect skin regeneration. Science. 1997;276(5309): Singer AJ and Clark RA. Cutaneous wound healing. N Engl J Med. 1999;341(10): Strodtbeck F. Physiology of wound healing. Newborn Infant Nurs Rev. 2001;14(2): Velnar T, Bailey T, Smrkolj V. The wound healing process: an overview of the cellular and molecular mechanisms. J Int Med Res. 2009;37(5): Hanks J and Spodnick G. Wound healing in the veterinary rehabilitation patient. Vet Clin North Am Small Anim Pract. 2005;35(6): Thomas DW, O'Neill ID, Harding KG, et al. Cutaneous wound healing: a current perspective. J Oral Maxillofac Surg. 1995;53(4):

52 8. Guo S and DiPietro LA. Factor affecting wound healing. J Dent Res. 2010;89(3): Lazarus GS, Cooper DM, Knighton DR, et al. Definitions and guidelines for assessment of wounds and evaluation of healing. Wound Repair Regen. 1994;2(3): Schreml S, Szeimies RM, Prantl L, et al. Wound healing in the 21st century. J Am Acad Dermatol. 2010;63(5): Deuel TF, Kawahara RS, Mustoe TA, et al. Growth factors and wound healing: platelet-derived growth factor as a model cytokine. Annu Rev Med. 1991;42(1): Barrientos S, Stojadinovic O, Golinko MS. Growth factors and cytokines in wound healing. Wound Repair Regen. 2008;16(5): Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol. 2007;25(1): Monaco JL and Lawrence WT. Acute wound healing an overview. Clin Plast Surg. 2003;30(1): Hosgood G. Stages of wound healing and their clinical relevance. Vet Clin North Am Small Anim Pract. 2006;36(4): Dahlbäck B. Blood coagulation. Lancet. 2000;355(5): Witte MB and Barbul A. General principles of wound healing. Surg Clin North Am. 1997;2(2): Robson MC, Steed DL, Franz MG. Wound healing: biologic features and approaches to maximize healing trajectories. Curr Probl Surg. 2001;38(2): Broughton G, Janis JE, Attinger CE. Wound healing: an overview. Plast Reconstr Surg. 2006;117(7):1e-S-32e-S. 20. Schaffer MR, Tandry U, Gross SS, et al. Nitric oxide regulates wound healing. J Surg Res. 1996;63(2): Read FH. (1980). Electromagnetic radiation. Chichester, England; New York: John Wiley & Sons. 38

53 22. Bertolotti M. (2005). The history of the laser. Bristol: Institute of Physics Publishing. 23. Hecht J. A short history of laser development. Appl Opt. 2010;49(25): Svelto O and Hanna DC. (2010). Principles of lasers. New York: Springer. 25. Webert RL. (1980). Pioners of science: Nobel prize winners in physics. Bristol: American Institute of Physics, Bristol and London. 26. Rullière C. (2005). Femtosecond laser pulses: principles and experiments. New York: Springer. 27. Renk KF. (2012). Basics of laser physics: for students of science and engineering. New York: Springer. 28. Breck HC, Ewing J, Hecht F. (2012). Introduction to Laser Technology, Fourth Edition. Hoboken, New Jersey: John Wiley & Sons, Inc. 29. Hooker S. (2010). Laser physics. Oxford, New York: Oxford University Press. 30. Milonni PW. (2010). Lasers physics. Hoboken, New Jersey: John Wiley & Sons. 31. Andersen K. Laser Technology a Surgical Tool of the Past, Present, and Future. Aorn J. 2003;78(5): Chung H, Dai T, Sharma SK, et al. The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng. 2012;40(2): Yadav RK. Definitions in laser technology. J Cutan Aesthet Surg. 2009;2(1): Williams D. Laser basics. Anaesth & Intensive care med. 2008;9(12): Hashmi JT, Huang YY, Sharma SK, et al. Effect of pulsing in low-level light therapy. Lasers Surg Med. 2010;42(6): da Silva JP, da Silva MA, Almeida AP, et al. Laser therapy in the tissue repair process: a literature review. Photomed Laser Surg. 2010;28(1): Andrade Fdo S, Clark RM, Ferreira ML. Effects of low-level laser therapy on wound healing. Rev Col Bras Cir. 2014;41(2): Saltmarche AE. Low-level laser therapy for healing acute and chronic wounds - the extendicare experience. Int Wound J. 2008;5(2): Lins RD, Dantas EM, Lucena KC, et al. Biostimulation effects of low-power laser in 39

54 the repair process. An Bras Dermatol. 2010;85(6): Hawkins D, Houreld N, Abrahamse H. Low-level laser therapy (LLLT) as an effective therapeutic modality for delayed wound healing. Ann N Y Acad Sci. 2005;1056(1): Hopkins JT, McLoda TA, Seegmiller JG, et al. Low-Level laser therapy facilitates superficial wound healing in humans: A triple-blind, sham-controlled study. J Athl Train. 2004;39(3): Demidova-Rice TN, Salomatina EV, Yaroslavsky AN, et al. Low-level light stimulates excisional wound healing in mice. Lasers Surg Med. 2007;39(9): Adamskaya N, Dungel P, Mittermayr R, et al., Light therapy by blue LED improves wound healing in an excision model in rats. Injury. 2011;42(9): Prindeze NJ, Moffatt LT, Shupp JW. Mechanisms of action for light therapy: a review of molecular interactions. Exp Biol Med. 2012;237(11): Gao X and Xing D. Molecular mechanisms of cell proliferation induced by low power laser irradiation. J Biomed Sci. 2009;16(1): Wilson DF and Vinogradov SA. Mitochondrial Cytochrome c Oxidase: Mechanism of Action and Role in Regulating Oxidative Phosphorylation. J Appl Physiol. 2014;117(12): Schertl P and Braun HP. Respiratory electron transfer pathways in plant mitochondria. Front Plant Sci. 2014;5(3): Silveira PC, Streck EL, Pinho RA. Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy. J Photochem Photobiol B. 2007;86(3): Chen CH, Hung HS, Hsu SH. Low-energy laser irradiation increases endothelial cell proliferation, migration, and enos gene expression possibly via PI3K signal pathway. Lasers Surg Med. 2008;40(1): Peplow PV, Chung TY, Baxter GD. Laser photobiomodulation of wound healing: a review of experimental studies in mouse and rat animal models. Photomed Laser Surg. 2010;28(3): Huang YY, Chen AC, Carroll JD, et al. Biphasic dose response in low level light therapy. Dose Response. 2009;7(4): Huang YY, Sharma SK, Carroll J, et al. Biphasic dose response in low level light therapy - an update. Dose Response. 2011;9(4):

55 53. Colombo F, Neto Ade A, Sousa AP, et al. Effect of low-level laser therapy (λ660 nm) on angiogenesis in wound healing: a immunohistochemical study in a rodent model. Braz Dent J. 2013;24(4): Hawkins DH and Abrahamse H. The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation. Lasers Surg Med. 2006;38(1): Houreld NN and Abrahamse H. Laser light influences cellular viability and proliferation in diabetic-wounded fibroblast cells in a dose- and wavelengthdependent manner. Lasers Med Sci. 2008;23(1): Basso FG, Pansani TN, Turrioni AP, et al. In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts. Int J Dent. 2012;71(1): Meireles GC, Santos JN, Chagas PO, et al. Effectiveness of laser photobiomodulation at 660 or 780 nanometers on the repair of third-degree burns in diabetic rats. Photomed Laser Surg. 2008;26(1): Mester E, Spiry T, Szende B. Effects of laser rays on wound healing. Bull Soc Int Chir. 1973;32(2): Medrado AR, Pugliese LS, Reis SR, et al. Influence of low level laser therapy on wound healing and its biological action upon myofibroblasts. Lasers Surg Med. 2003;32(3): Mester E, Mester AF, Mester A. The biomedical effects of laser application. Lasers Surg Med. 1985;5(1): Posten W, Wrone DA, Dover JS, et al. Low-level laser therapy for wound healing: mechanism and efficacy. Dermatol Surg. 2005;31(3): Kajagar BM and Godhi AS. Efficacy of low level laser therapy on wound healing in patients with chronic diabetic foot ulcers a randomized control trial. Indian J Surg. 2012;74(5): Lundeberg T and Malm M. Low-power HeNe laser treatment of venous leg ulcers. Ann Plast Surg. 1991;27(6): Lagan KM, Clements BA, McDonough, et al. Slow intensity laser therapy (830nm) in the management of minor postsurgical wounds: a controlled clinical study. Lasers Surg Med. 2001;28(1): Souza NH, Ferrari RA, Silva DF, et al. Effect of low-level laser therapy on the modulation of the mitochondrial activity of macrophages. Braz J Phys Ther. 41

56 2014;18(4): Tacon KC, Santos HC, Parente LM, et al. Healing activity of laser InGaAlP (660nm) in rats. Acta Cir Bras. 2011;26(5): Busnardo VL and Biondo-Simões ML. [Effects of low-level helium-neon laser on induced wound healing in rats]. Rev Bras Fisioter. 2010;14(1): Lyons RF, Abergel RP, White RA, et al. Biostimulation of wound healing in vivo by a helium-neon laser. Ann Plast Surg. 1987;18(1): Vasilenko T, Slezák M, Kovác I, et al. The effect of equal daily dose achieved by different power densities of low-level laser therapy at 635 and 670 nm on wound tensile strength in rats: a short report. Photomed Laser Surg. 2010;28(2): Liu A, Moy RL, Ozog DM. Current method employed in the prevention and minimization of surgical scars. Dermatol Surg. 2011;37(12): Lim W, Choi H, Kim J, et al. Anti-inflammatory effect of 635 nm irradiations on in vitro direct/indirect irradiation model. J Oral Pathol Med. 2015;44(2): Hagiwara S, Iwasaka H, Hasegawa A, et al. Pre-Irradiation of blood by gallium aluminum arsenide (830 nm) low-level laser enhances peripheral endogenous opioid analgesia in rats. Anesth Analg. 2008;107(3): Karu T, Pyatibrat L, Kalendo G. Irradiation with He-Ne laser increases ATP level in cells cultivated in vitro. J Photochem Photobiol B. 1995;27(3): Sushko BS, Lymans'kyĭ IuP, Huliar SO. [Action of the red and infrared electromagnetic waves of light-emitting diodes on the behavioral manifestation of somatic pain]. Fiziol Zh. 2007;53(3): Ponnudurai RN, Zbuzek VK, Wu WH. Hypoalgesic effect of laser photobiostimulation shown by rat-tail flick test. Acupunct Electrother Res.1987;12(2): Rocha Júnior AM, Vieira BJ, de Andrade LC, et al. Low-level laser therapy increases transforming growth factor-beta2 expression and induces apoptosis of epithelial cells during the tissue repair process. Photomed Laser Surg. 2009;27(2): Gonçalves WL, Souza FM, Conti CL, et al. Influence of He-Ne laser therapy on the dynamics of wound healing in mice treated with anti-inflammatory drugs. Braz J Med Biol Res. 2007;40(6): Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes 42

57 small RNAs with antisense complementarity to lin-14. Cell. 1993;75(5): Ketting RF. MicroRNA biogenesis and function: an overview. Adv Exp Med Biol. 2011;70(4): Viticchiè G, Lena AM, Cianfarani F, et al. MicroRNA-203 contributes to skin reepithelialization. Cell Death Dis. 2012;29(3): Banerjee J and Sen CK. MicroRNAs in skin and wound healing. Methods Mol Biol. 2013;93(6): Banerjee J, Chan YC, Sen CK. MicroRNAs in skin and wound healing. Physiol Genomics. 2011;43(10): Bartel DP. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 2004;116(2): Schneider MR. MicroRNAs as novel players in skin development, homeostasis and disease. Br J Dermatol. 2012;166(1): Yi R, O'Carroll D, Pasolli HA, et al. Morphogenesis in skin is governed by discrete sets of differentially expressed micrornas. Nat Genet. 2006;38(3): Gu X, Nylander E, Coates PJ, et al. Effect of narrow-band ultraviolet B phototherapy on p63 and microrna (mir-21 and mir-125b) expression in psoriatic epidermis. Acta Derm Venereol. 2011;91(4): Sand M, Gambichler T, Sand D, et al. MicroRNAs and the skin: tiny players in the body's largest organ. J Dermatol Sci. 2009;53(3): Li P, He Q, Luo C, et al. Differentially expressed mirnas in acute wound healing of the skin: a pilot study. Medicine. 2015;94(7): Li D, Li X, Wang A, et al. MicroRNA-31 Promotes Skin Wound Healing by Enhancing Keratinocyte Proliferation and Migration. J Invest Dermatol. 2015; doi: /jid Shilo S, Roy S, Khanna S, et al. MicroRNA in cutaneous wound healing: a new paradigm. DNA Cell Biol. 2007;26(4): Liu G, Friggeri A, Yang Y, et al. mir-147, a microrna that is induced upon Tolllike receptor stimulation, regulates murine macrophage inflammatory responses. Proc Natl Acad Sci U S A. 2009;106(37): Lai WF and Siu PM. MicroRNAs as regulators of cutaneous wound healing. J Biosci. 2014;39(3):

58 93. Yang X, Wang J, Guo SL, et al. mir-21 promotes keratinocyte migration and reepithelialization during wound healing. Int J Biol Sci. 2011;7(5): Wang T, Feng Y, Sun H, et al. mir-21 regulates skin wound healing by targeting multiple aspects of the healing process. Am J Pathol. 2012;181(6): van Solingen C, Araldi E, Chamorro-Jorganes A, et al. Improved repair of dermal wounds in mice lacking microrna-155. J Cell Mol Med. 2014;18(6): Kushibiki T, Hirasawa T, Okawa S, et al. Regulation of mirna Expression by Low- Level Laser Therapy (LLLT) and Photodynamic Therapy (PDT). Int J Mol Sci. 2013;14(7): Wang J, Huang W, Wu Y, et al. MicroRNA-193 pro-proliferation effects for bone mesenchymal stem cells after low-level laser irradiation treatment through inhibitor of growth family, member 5. Stem Cells Dev. 2012;21(13): Kurach LM. The effect of Low-Level Laser Therapy on the Healing of Open Wounds in Dogs. Michigan Veterinary Conference 2015 Abstract. 44

59 CHAPTER II Introduction to cell culture experiments Cell culture models represent useful tools with which individual cellular properties and behaviors as well as basic biological processes can be readily evaluated. In this project, some aspects of the safety and efficacy of laser therapy were evaluated using canine epidermal keratinocyte progenitors (CPEK) in a wound-healing model, where cellular migration and proliferation were assessed. To date, the effect of LLLT using a canine cell culture model has never been evaluated. This type of study represents an informative and non-invasive approach to understanding the basic mechanisms that are likely to be altered during LLLT. 2.1 Scratch migration and proliferation assays The scratch migration and cell proliferation WST-1 assays have both been employed frequently in other in vitro wound healing experiment models [1-3]. The scratch migration assay was selected for this study, as it is a straightforward, relatively inexpensive, reproducible, and well-defined method to measure basic cellular migration parameters over time [2]. It represents a widely accepted method for measuring and evaluating cellular migration in vitro and it models many aspects of in vivo cellular migration [3]. To evaluate and compare cellular proliferation among all groups over time, the ready-to-use cell proliferation reagent WST-1 was employed. This reagent provides a simple and appropriate method to measure cellular metabolic activity, which is based on the cleavage of the tetrazolium salt by cellular mitochondrial dehydrogenases to soluble, 45

60 non-cytotoxic, highly colored end product called formazans [4,5]. An increase in the number of viable cells results in higher mitochondrial enzymatic activity (mitochondrial numbers are relatively constant on a per cell basis), and leads to a greater amount of formazan dye production [4]. The formazan dye is then quantified by determining the light absorbance at the wavelength of maximal absorbance (450 nm) [4]. The quantity of formazan dye produced is directly proportional to the number of metabolically active cells in the culture medium [4,5]. The method is considered more rapid and more sensitive than those using other tetrazolium compounds MTT-, XTT-, or MTS [5]. 2.2 Pilot study In the first part of this project, a pilot study was performed to examine different incubation protocols, laser doses and other specific parameters of the study design for both the scratch migration assay and proliferation assay. First, different incubation protocols looking at the optimal time the plated cells should be maintained in 10 % serum medium and then serum deprived prior to laser therapy were carried. Seeding the cells in 10% serum medium for 24 hours, then depriving them for a 12-hour period, showed the best cell survival time and lowest risk of contamination over the entire culture period. Using this study protocol, several laser doses ranging from 0.1 J/cm 2 to 15 J/cm 2 were then tested and compared for both assays. Low-level laser therapy treatment protocols available in the literature typically employ wavelengths between 500 and 1000 nm and doses of less than 4 J/cm 2, but the optimal laser doses are still essentially unknown. The tested laser dosages provided a range of results in terms of proliferation rate and migration characteristics and were compared and considered to arrive at the dosages used 46

61 in the final experiments. Finally, the optimal number of cells to plate for each analysis was determined by performing the experiments at different cellular concentrations and according to other similar protocols available in the literature. 2.3 References 1. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005;11(3): Cory G. Scratch-wound assay. Methods Mol Biol. 2011;769(4): Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2(2): Kumarasuriyar A. Cell proliferation Reagent WST-1 from Roche applied science Accessed 16 March Reviews/40930-Cell-ProliferationReagent WST-1-From-Roche-Applied-Science/. 5. Ishiyama M, Tominaga H, Shiga M, et al. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull. 1996;19(3):

62 CHAPTER III An in vitro method to test the safety and efficacy of low-level laser therapy (LLLT) in the healing of a canine skin model Dominique Gagnon 1, Thomas W. Gibson 1, Ameet Singh 1, Alex zur Linden 1 ; Jaimie E. Kazienko 2 ; Jonathan LaMarre 2 1 Department of Clinical Studies, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Ontario, N1G 2W1, Canada 2 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, 50 Stone Road East, Ontario, N1G 2W1, Canada Corresponding author: Dominique Gagnon. Ontario Veterinary College (OVC), University of Guelph, 50 Stone Road East, Ontario, N1G 2W1, Canada Phone: Fax: dgagno01@uoguelph.ca 48

63 3.1 Abstract Background Low-level laser therapy (LLLT) has been used clinically as a treatment modality for a variety of medical conditions including wound-healing processes. It is an attractive and emerging method to enhance wound healing and improve clinical outcomes both in human and veterinary medicine. Despite the fact that the use of LLLT continues to gain in popularity, there is no universally accepted theory that defends all its cellular effects and beneficial biological processes in tissue repair. The present study was designed to evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK) in an in vitro wound healing model. Results Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. Fifteen independent replicates were performed for each assay. Canine epidermal keratinocyte cells exposed to LLLT with 0.1, 0.2, and 1.2 J/cm 2 migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < ) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm 2. Irradiation with 10 J/cm 2 was characterized by decreased cellular migration and proliferation. These results revealed that low-level laser therapy has a measurable, dosedependent effect on two different aspects of keratinocyte biology in vitro. Conclusion In this in vitro wound-healing model, LLLT increased cellular migration and proliferation at doses of 0.1, 0.2, and 1.2 J/cm 2 while exposure to 10 J/cm 2 decreased 49

64 cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior. Keywords Canine epidermal keratinocytes progenitors; low-level laser therapy; proliferation assay; scratch migration assay; wound healing. 3.2 Background The healing of skin wounds represents an ongoing challenge in both human and veterinary medicine [1,2]. The care and management of both acute and chronic cutaneous wounds can be time consuming and may have significant health and economic consequences [2]. An emerging treatment modality involves the use of laser therapy, further described as low-level laser therapy (LLLT), which has been postulated to accelerate healing of damaged tissues and improve clinical outcomes in several independent studies [3,4]. Despite the positive results demonstrated in previous reports, the effectiveness and underlying mechanisms of LLLT remain largely speculative and unresolved [4]. Wound healing was one of the first clinical applications of laser therapy following its discovery by Endre Mester in 1967 [5]. It has been suggested that the use of this treatment modality on cutaneous wounds provides several benefits, including pain control, reduction of inflammation, modulation of the immune system, and acceleration of wound healing [6-8]. In recent years, the medical applications of LLLT in human medicine have expanded to include the treatment of many conditions such as neurological processes, musculoskeletal injuries, soft tissue injury, abscesses, dental disease, and 50

65 various dermatologic ailments [8,9]. Low-level laser therapy causes low or imperceptible temperature changes to the treatment area [10]. At the cellular level, the effect of light therapy is thought to be secondary to the absorption of red and near-infrared light by cytochrome c oxidase, improving electron transport, production of adenosine triphosphate (ATP), release of nitric oxide, and the modulation of reactive oxygen species [10,11]. Although LLLT is believed to result in increased cellular metabolism via activation of the cellular respiratory chain, the exact mechanism of action at the cellular level remains unknown [11,12]. Lowlevel laser therapy as a treatment modality remains controversial due to an incomplete understanding of the underlying mechanism(s) responsible for the observed beneficial effects, and the large number of different irradiation protocols found in the literature, making comparisons between studies challenging [10-12]. For these reasons, light therapy remains an alternative treatment and its use is largely empirical [8,11]. Numerous reports in the literature have shown beneficial results of LLLT on the rate and extent of wound healing using various models [3,12]. The effect of laser therapy on in vitro cellular migration and proliferation of canine skin culture models has not been studied to date. The objective of this study was to evaluate both migration and proliferation of canine epidermal keratinocytes after different exposures/doses of LLLT in an in vitro wound-healing model. 51

66 3.3 Methods Cell culture Canine epidermal keratinocyte progenitors (CPEK) were purchased from CELLnTEC Advance Cell Systems. These cells are cryo-preserved after isolation from normal tissue and have not undergone any transformation events. The CnT-09 medium, provided with the cell lines, was used for all CPEK canine epidermal cell cultures. CnT- 09 is a liquid medium package including both basal medium (CnT-BM.2, 500 ml) and separate supplements (A [10 % fetal bovine serum (FBS), 50 ml) and B [L-glutamine, 5 ml]). The supplements can be used at different concentrations, depending on the volume added to the basal medium. For this study, a concentration of 10% and 1% (serumdeprived medium) were used. All canine epidermal keratinocyte cells were cultured in 10 ml of 10% serum medium (CnT-09) in 75 cm 3 tissue culture flasks and incubated at 37 C in an atmosphere of 5% CO 2 and 95% air and passaged between 50% and 90% confluence. The medium was changed every forty-eight hours and replaced in the humidified incubator until an adequate numbers of cells were obtained for each experiment. For the scratch migration and proliferation assays, CPEK cells were grown to approximately 90% confluency and 40% confluency, respectively. Third- to fifth-passage cells were used for all experiments Study protocols Cells in T-75 flasks were trypsinized and plated on sterile 6-well tissue culture plates (Corning). For the scratch migration assays, the cells were seeded at 1 x 10 6 cells per well in 2 ml keratinocyte growth medium CnT-09. For the proliferation assays, cells 52

67 were seeded at a concentration of 4 x 10 4 cells per well. For both experiments, plates were incubated for twenty-four hours under the conditions previously mentioned. Following this incubation, the medium of each well was replaced with 2 ml of low serum (1%) medium, and incubated for an additional twelve hours. The medium of each well was then removed and replaced with phosphate buffered saline (PBS). For the scratch migration assay, a sterile p200 pipette tip was used to make a linear disruption of the monolayer of cells, simulating a wound-healing model. The scratch was created vertically in the middle of each well. The detached cells secondary to the creation of the scratch were carefully removed by rinsing each well with 1mL of PBS. Formation of the in vitro wound was confirmed by inverted light microscopy. To ensure that the same field was identified during subsequent image acquisition, two vertical lines on each side of the scratch and one horizontal line, separating the wound in half, were placed with an indelible marker on the outside bottom of each well. These markings served as reference points for photographic documentation Experimental design A randomized, double blinded and controlled study design was used to evaluate the effect of LLLT delivered at different doses over time on canine skin culture. Cellular migration and proliferation were determined using the scratch migration assay and proliferation assay, respectively. Six groups composed of two different controls (positive and negative) and four treatments were compared for the scratch migration assay; five groups composed of one control (negative) and the same four-irradiation treatments were evaluated in the proliferation assay. A summary of the selected laser parameters for each 53

68 group is provided (Table 1). The energy density (J/cm 2 ) for all treatment groups was calculated by multiplying the exposure time (s) by the power output of the laser, divided by the surface area (cm 2 ). The surface area exposed selected for the study was identical to the surface area of each culture well on a 6-well plate (9.62cm 2 ). Surface area (cm 2 ) = πr 2 Irradiance (J/cm 2 ) = Time (s) x Power (W) Surface area (cm 2 ) Randomization of each group on the plate was achieved using a random number table provided in a statistics textbook [13]. The smallest number obtained represented the first well position, and then in ascending order, each group was assigned a position commencing with control positive, control negative, treatment 1, treatment 2, treatment 3, and treatment 4. Each experiment was run in quintuplicate using cells between the thirdand fifth-passage. A total of fifteen independent replicates were performed for both assays. For the proliferation assay, three plates were made per passage to allow cellular proliferation quantification at three different time points Low-level laser therapy Laser therapy was carried out using a high-power Helium-Neon (He-Ne) Class IV laser system. Laser safety guidelines were followed as recommended by the manufacturer. Cells cultured in 6-well plates as described were irradiated using the large conical laser head provided. The handpiece was suspended from a custom-made stand holder and laser irradiation was carried at a fixed distance of five centimeters by measuring the distance between the laser head and the plate. This distance was 54

69 specifically chosen as it provided uniform irradiation of the surface of each well. Laser irradiation was performed perpendicular to the bottom of the culture well, once, using continuous emission at a wavelength of 650 nm. Laser irradiation was performed in the same manner each time. The treated groups (treatment 1 to 4) were irradiated with one of the four-energy densities tested, as described above. The wells assigned to control groups (positive and negative controls) were sham-irradiated; they were maintained under the laser head for the minimum irradiation time used in the irradiated groups, without activating the laser source. During LLLT, the non-irradiated wells were covered using a clean, double-folded, white, commercial paper towel to prevent incidental irradiation. Following laser irradiation, 2 ml of normal growth medium (10% serum medium) was added to the well of the control positive, and 2 ml of low-serum medium (1% serum) was added to the control negative and all four treated groups Scratch migration assay A scratch was created in a cell monolayer as described above, which simulated a wound. The change in the wound surface area was compared among groups over time. Digital photographic images were obtained at the 0-, 12-, 24-, 36- and 48-hour time points (or until complete closure of the scratch wound was observed) using a motorized inverted microscope. Between each time point, the plates were incubated under the conditions described above. Following the acquisition of all images, the surface area of each scratch was measured and outlined by two independent observers (blinded to the treatment group) using the most recent Adobe Photoshop CC software. The surface area of each wounded region of the cell monolayer was then transformed into a square of equal surface area, and 55

70 the linear mean length of each square was compared among groups over time. The rate of closure was quantified and compared between all groups for statistical analysis Proliferation assay To evaluate and compare cellular proliferation among all groups over time, the ready-to-use cell proliferation reagent, water-soluble tetrazolium (WST-1) was used. At the time of the experiment, 1.5 ml of medium was removed from each well, leaving a volume of 0.5 ml over the seeded cells. Fifty microliters of the cell proliferation reagent WST-1 was then added to each well of the 6-well acrylic plate, gently mixed with the medium for 30 seconds, and the plate was incubated for fifteen minutes under the conditions previously described. Three 100 µl aliquots from each well were transferred to a sterile 96-well spectrophotometer plate. The quantity of formazan dye produced by cellular metabolism was quantified by measuring its absorbance at a wavelength at 450 nm using a microplate reader at 0-, 24- and 48-hour time points. The mean absorbance obtained at each time point was compared between all groups for statistical significance. 3.4 Statistical analysis For statistical analysis, the effect of LLLT delivered at various doses over time on canine skin culture was evaluated. For both the scratch migration assay and proliferation assay, a statistical program (SAS 9.2) was used to fit a general linear mixed model. The design was a two-factor factorial in a randomized complete block design (RCBD) with fixed-effect factors treatment and time. To accommodate time being a repeated measure in the scratch migration assay, the following correlation structures (offered by SAS) were 56

71 attempted: ar(1), arh(1), toep, toep(2), toep(3), toeph, toeph(2), toeph(3), un, un(2), and un(3). The random blocking effect was plates nested within cohorts. In the proliferation assay, time was not a repeated measure as destructive sampling occurred over time. To accommodate subsampling, a random effect of treatment by time by plates nested within cohorts was included in that model. Among the error structures that converged, the one with the smallest Akaike Information Criterion (AIC) was chosen. A two-way interaction (time and treatment) was considered. However, if terms were not significant, they were removed from the model. Differences were considered significant at P To assess the ANOVA assumptions, comprehensive residual analyses were performed. The assumption of normality was formally tested by use of Shapiro-Wilk, Kolmogorov- Smirnov, Cramér-von Mises, and Anderson-Darling tests. Residuals were plotted against the predicted values and explanatory variables were used in the models to show outliers, bimodal distributions, the need for data transformations, or other issues that should be addressed. 3.5 Results Scratch migration assay A total of fifteen experiments were included in the study. To attempt to meet the ANOVA assumptions of the statistical analysis, a square-root transformation was applied to all data as well as accommodating unequal variance in time (the error structure chosen was arh [1]). The results obtained at the 48- and 60-hour time points were removed from the analysis as most of the groups tested were closed, except for the vast majority of cells irradiated at a higher dose of 10 J/cm 2. No apparent outliers were identified in the 57

72 analysis. There was no significant inter-observer variability observed for each surface area measured; all of the effects involving the observers had a p > The linear mean length of each wounded region of the cell monolayer was compared among groups over time. There was no statistical difference (p > 0.05) in the linear mean length of each scratch created in the cell monolayer at the beginning of the study period (Figure 1). Twelve hours following laser therapy (figure 2), there was a significant reduction in the linear mean length of the control positive (p < ) and the cells irradiated with 0.1 J/cm 2 (p = ), 0.2 J/cm 2 (p = ) and 1.2 J/cm 2 (p = 0.026) compared to the cells irradiated with 10 J/cm 2. A significant diminution of the area was also noted between the control positive (p < ) and the cells irradiated with the two lower energy doses (p < 0.01) compared to the control negative, and between the control positive and the cells irradiated with 1.2 J/cm 2 (p = ). Twenty-four hours (Figure 3) and 36 hours (Figure 4) following irradiation therapy, there was no significant difference observed in the linear mean length of the control positive wounded region compared to the groups irradiated with 0.1, 0.2, and 1.2 J/cm 2 (p > 0.15). The positive control and all three groups with lower irradiation did, however, show a significantly reduced linear mean length compared to the control negative (p < 0.03) and the group irradiated with 10 J/cm 2 (p < ). A significant difference was also identified between the control negative and the cells irradiated with 10 J/cm 2 (p < 0.003) at both time points. For all 15 experiments, the time of closure among groups was similar and followed a predictable pattern. The cells irradiated with the highest dose of 10 J/cm 2 consistently showed the slowest closure rate compared to all other groups. Overall, the 58

73 results of the scratch migration assay revealed that wounded canine epidermal keratinocytes closed earlier in the non-irradiated cultures maintained in 10% serum medium (positive control) and in cultures exposed to a single dose of 0.1, 0.2 or 1.2 J/cm 2 compared to the non-irradiated cells cultured in serum-reduced medium (negative control) and the cells irradiated with 10 J/cm Proliferation Assay A total of fifteen experiments, from cell passages three to five, were included in the study. Three replicated plates were used for each experiment to evaluate cellular proliferation over time. An outlier was identified in the proliferation assay but was retained in the analysis. The assumption of normality was mildly violated because of that one outlier; otherwise the assumption of the ANOVA analysis was adequately met. No transformation was required. The number of cells at each time point was assessed by comparing the mean absorbance of the formazan dye produced by cellular metabolism following the addition of the cell proliferation reagent WST-1. There was no statistical difference (p > 0.5) in the mean absorbance among groups at the beginning of the study period (Figure 5). Twenty-four hours after laser irradiation, the mean absorbance of the cells irradiated with 0.1, 0.2, and 1.2 J/cm 2 was significantly increased compared to the non-irradiated cells cultured in serum-deprived medium (p < ) and the cells irradiated with 10 J/cm 2 (p < ). There was no significant difference between the control group and the cells irradiated with 10 J/cm 2 at that time point (p = ) (figure 6). Forty-eight hours after irradiation, the mean absorbance of WST-1 containing media over canine epidermal 59

74 keratinocytes irradiated with 0.1, 0.2, or 1.2 J/cm 2 was significantly higher than the control cultures (p < ) and the cultures irradiated with 10 J/cm 2 (p < ). In addition, cultures irradiated with 0.1 J/cm 2 (p = 0.001) or 0.2 J/cm 2 (p = ) had significantly greater cellular proliferation compared to the cells irradiated with 1.2 J/cm 2. There were no significant differences in cellular proliferation between the two lower energy doses (p = ), nor between the control negative group and the cells irradiated with 10 J/cm 2 (p = ) at the 48-hour time point (figure 7). The results of the cell proliferation assay indicate that canine epidermal keratinocytes exposed to a single dose of 0.1, 0.2 or 1.2 J/cm 2 proliferated more rapidly than non-irradiated cells cultured under serum-reduced conditions and cultures irradiated with 10 J/cm Discussion The present study has evaluated the effects of LLLT on in vitro responses in a model of wound healing using cultured canine epidermal keratinocytes. To the authors knowledge, these represent the first studies of this kind in veterinary medicine. Irradiation therapy was delivered as a single exposure from a high-power He-Ne Class IV laser unit system (650nm). Cellular migration and proliferation, which are both essential in the normal wound healing process in vivo, were evaluated. The scratch migration and cell proliferation WST-1 assays have both been employed frequently in other wound healing experiments in the literature [14-16]. The scratch migration assay was selected for this study, as it is a straightforward, relatively inexpensive, reproducible, and well-defined method to measure basic cellular migration 60

75 parameters over time [15]. It represents a widely accepted method for measuring and evaluating cellular migration in vitro and it models many aspects of in vivo cellular migration [16]. To evaluate and compare cellular proliferation among all groups over time, the ready-to-use cell proliferation reagent WST-1 was employed. This reagent provides a simple and accurate method to measure cellular metabolic activity, which is based on the cleavage of the tetrazolium salt by cellular mitochondrial dehydrogenases to soluble, non-cytotoxic, highly colored end product, called formazans [17]. An increase in the number of viable cells results in higher mitochondrial dehydrogenase activity (mitochondrial numbers are relatively constant on a per cell basis), which leads to a greater amount of formazan dye production [17]. The formazan dye is then quantified by measuring the light absorbance at the wavelength of maximal absorbance (450 nm) [17]. The quantity of formazan dye is directly proportional to the number of metabolically active cells in the culture medium [17]. The method is considered more rapid and more sensitive than those using other tetrazolium compounds MTT-, XTT-, or MTS [18]. The results of both the scratch migration and proliferation assays showed that canine epidermal keratinocytes produce a measurable biological response in vitro that is likely favorable for wound healing when exposed to low doses of LLLT (0.1, 0.2 or 1.2 J/cm 2), compared to non-irradiated cultures. Overall, the positive control group in which normal serum, containing numerous additional growth factors and nutrients, showed the fastest rate of closure, which was expected. This rate of closure was, however, not significantly different compared to the cells maintained in serum-deprived medium and exposed to low-levels of laser irradiation. Because a cell proliferation reagent was added directly to the medium in the cell proliferation assay, a positive control was not included 61

76 as the metabolic consequences of the high serum concentration that is present alters the spectral properties of the medium. Significant stimulatory effects of LLLT on cell migration and proliferation of canine keratinocytes was observed. These results are in agreement with other experimental analyses that report a beneficial effect of laser irradiation therapy in wound healing [3,12] and help identify the individual processes that are influenced in keratinocytes. Importantly, LLLT at the highest exposure dosage (10J/cm 2 ) resulted in an inhibitory effect, suggesting that dose level is extremely important in the overall biologic response. Dose-responses of this type have been previously described in several in vivo clinical experiments, animal models, and cell cultures [11,19,20]. The Arndt-Schulz Law is commonly cited as an appropriate model to demonstrate the dose-dependent effect of laser light therapy and suggests that low levels of light have a better effect in wound healing than higher levels, which may have an inhibitory or cytotoxic effect [19],[20]. In their work to establish the behavior in vitro of human skin fibroblasts, Hawkins and Abrahamse (2006) used a He-Ne laser (632.8 nm) at different irradiation doses of 0.5, 2.5, 5, 10, and 16 J/cm 2. They demonstrated that higher laser doses (10 and 16 J/cm 2 ) resulted in increased cellular damage as well as decreased cellular viability and proliferation [21]. Houreld and Abrahamse (2008) used He-Ne (632.8nm), Gallium-Aluminum-Arsenide (GaAlAs (830nm)), and Neodymium Yttrium Aluminum Garnet (1064nm) lasers for treatment of in vitro wounded diabetic-induced fibroblasts irradiated with either 5 or 16 J/cm 2. Regardless of the wavelength used, all cells irradiated with 16 J/cm 2 showed incomplete wound closure, increased apoptosis, and decreased basic fibroblast growth factor expression. The fibroblasts responded better overall when irradiated with an 62

77 energy density of 5 J/cm 2 at a wavelength of nm [22]. Similarly, Basso et al. (2012) demonstrated that irradiation with Indium Gallium Arsenide Phosphide diode laser (780nm) of cultured human gingival fibroblasts with energy doses of 0.5 and 3 J/cm 2 resulted in a significant increase in cellular metabolism compared with the non-irradiated control group and the cells irradiated with higher energy doses of 5 and 7 J/cm 2 [23]. Limited animal model studies have also shown similar dose-dependent responses, leading to discrepancies in clinical outcomes observed. In their work on wound healing, Demidova-Rice and collaborators (2007) documented this phenomenon when they tested the effect of 635 nm non-coherent light at 1, 2, 10 and 50 J/cm 2 on full-thickness dorsal excisional wounds in mice. Comparison of the area under the healing curves generated based on the wound healing rates over time among groups revealed that a single exposure to 1, 2 and 10 J/cm 2 improved wound healing compared to the non-irradiated control group, while the group irradiated with 50 J/cm 2 had an inhibitory effect [11]. The concept of biphasic dose-response is important in radiation light therapy and the complexity of choosing amongst a large number of illumination parameters for each treatment may explain why there is disagreement among clinical results and lack of clear conclusions regarding the observed effects of LLLT [19,23]. In addition, Karu (1989) proposed that the magnitude of the laser photostimulation effect was dependent of the physiological state of the cell at the time of irradiation. Stressed, damaged, or poorly growing cultures respond overall better to the positive effect of laser therapy [24]. Karu s statement may also explain why the effect is not always detectable and why there are conflicting results found in the literature. Similar to other cell culture studies, the present analysis demonstrated that canine epidermal keratinocyte cultures irradiated with the highest dose 63

78 (10 J/cm 2 ) had the slowest in vitro wound closure rate and proliferation rate compared to cells irradiated with lower energy doses of 0.1, 0.2, and 1.2 J/cm 2. Overall, these results clearly show that LLLT can stimulate or impede cellular processes in a dose-dependent manner. The exact mechanisms by which laser therapy influences cellular processes remain controversial [10-12]. At the cellular level, the effect of LLLT is thought to be absorption of red and near infrared light by the photoreceptor cytochrome c oxidase located in mitochondria [10,11]. Few experiments have shown that cellular respiration is increased when mitochondria are exposed to light in the red and near-infrared spectral range [25-27]. Of them, Chen et al. (2008) demonstrated, in their study on endothelial cells, that low-energy laser irradiation at the cellular level increases endothelial cell proliferation, migration, and endothelial nitric oxidase synthetase (enos) protein expression through activation of the PI3K/Akt pathway [25]. Huang and colleagues (2013) provided evidence that low-power laser irradiation using a HeNe laser at a wavelength of 633nm and dose of 1.2 J/cm 2 induced nuclear redistribution and transcriptional activity of estrogen receptors, through activation of the PI3K/Akt signaling cascade, which may control cellular processes and regulation of gene expression [26]. Silveira et al. (2006) successfully demonstrated by mitochondrial enzyme evaluation that red and near-infrared light delivered for 10 days to iatrogenic wounds created on adult male Wistar rats significantly increased the activities of the respiratory chain enzyme complexes II and IV (cytochrome c oxidase). The instrument used in this study was a GaAlAs laser with a wavelength of 904nm [27]. The results of these analyses are in agreement with other data from the literature where cytochrome c oxidase is thought to be an important 64

79 photoreceptor of light in the red to near-infrared spectrum [10,12,25-27]. However, more studies are required to fully evaluate mitochondrial enzyme activities following LLLT and to understand its mechanisms of action at the cellular level. 3.7 Conclusion In conclusion, this study has provided novel findings using a previously-untested cell type concerning the biological effects of LLLT on important cellular parameters associated with wound healing in cultured canine epidermal keratinocytes. It is the first known study in veterinary medicine to evaluate the efficacy of LLLT in vitro. Cell culture models represent useful tools to evaluate the efficacy and safety of laser irradiation during wound healing, and are strongly indicated prior to the application of this treatment modality in a clinical setting. The findings of the study demonstrated that a low-level of laser irradiation delivered as a single dose caused increased migration and proliferation of canine epidermal keratinocytes compared to non-irradiated cells cultured in the same low serum medium. The results are supportive of previous cell culture studies in which acceleration of the healing process was observed following LLLT. Low-level laser therapy may stimulate or impede cellular processes in a dose-dependent manner, suggesting that appropriate protocol selection will be critical for improved clinical outcomes. It is clearly one responsibility of veterinarians to substantiate or refute the reported beneficial effects associated with the use of LLLT in our companion animal patients. Further in vitro and in vivo studies will be required to fully investigate the effects of LLLT on canine wound healing and the mechanisms by which this treatment mediates its effects so that continued improvements can be made. 65

80 3.8 List of abbreviations CPEK He-Ne LLLT WST-1 Canine epidermal keratinocyte progenitors Helium-Neon Low-level laser therapy Water-soluble tetrazolium 3.9 Competing interests The authors declare that they have no competing interests Authors contributions All authors approved the manuscript. DG, TG, AS, AZ, JK, and JL contributed to study design and data interpretation. DG was the principal investigator. A statistician, William Sears, performed all data analysis and results interpretation. DG wrote the manuscript and produced all figures. All authors read, corrected, and approved the final manuscript prior to submission Acknowledgements The research project was funded by the Ontario Veterinary College Pet Trust and equipment support was provided by LiteCure, Companion Therapy Laser System. These groups had no input or participation in preparing this manuscript. 66

81 3.12 References 1. Velnar T, Bailey T, Smrkolj V. The wound healing process: an overview of the cellular and molecular mechanisms. J Int Med Res. 2009;37(5): Saltmarche AE. Low-level laser therapy for healing acute and chronic wounds - the extendicare experience. Int Wound J. 2008;5(2): Kajagar BM and Godhi AS. Efficacy of low level laser therapy on wound healing in patients with chronic diabetic foot ulcers a randomized control trial. Indian J Surg. 2012;74(5): Prindeze NJ, Moffatt LT, Shupp JW. Mechanisms of action for light therapy: a review of molecular interactions. Exp Biol Med. 2012;237(11): Chung H, Dai T, Sharma SK, et al. The nuts and bolts of low-level laser (light) therapy. Ann Biomed Eng. 2012;40(2): da Silva JP, da Silva MA, Almeida AP, et al. Laser therapy in the tissue repair process: a literature review. Photomed Laser Surg. 2010;28(1): Medrado AR, Pugliese LS, Reis SR, et al. Influence of low level laser therapy on wound healing and its biological action upon myofibroblasts. Lasers Surg Med. 2003;32(3): Peplow PV, Chung TY, Ryan B, et al. Laser photobiomodulation of gene expression and release of growth factors and cytokines from cells in culture: a review of human and animal studies. Photomed Laser Surg. 2010;28(3): Lins RD, Dantas EM, Lucena KC, et al. Biostimulation effects of low-power laser in the repair process. An Bras Dermatol. 2010;85(6): Hawkins D, Houreld N, Abrahamse H. Low level laser therapy (LLLT) as an effective therapeutic modality for delayed wound healing. Ann N Y Acad Sci. 2005;1056(1): Demidova-Rice TN, Salomatina EV, Yaroslavsky AN, et al. Low-level light stimulates excisional wound healing in mice. Lasers Surg Med. 2007;39(9): Hopkins JT, McLoda TA, Seegmiller JG, et al. Low-Level laser therapy facilitates superficial wound healing in humans: A triple-blind, sham-controlled study. J Athl Train. 2004;39(3):

82 13. Hulley SB, Cummings SR, Browner WS, et al. (2007). Designing Clinical Research, 3rd Edition. 3rd ed. Philadelphia: Lippincott Williams & Wilkins. 14. Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev. 2005;11(3): Cory G. Scratch-wound assay. Methods Mol Biol. 2011;769(4): Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2(2): Kumarasuriyar A. Cell proliferation Reagent WST-1 from Roche applied science Accessed 16 March WST-1-From-Roche-Applied-Science/. 18. Ishiyama M, Tominaga H, Shiga M, et al. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull. 1996;19(3): Huang YY, Chen AC, Carroll JD, et al. Biphasic dose response in low level light therapy. Dose Response. 2009;7(4): Huang YY, Sharma SK, Carroll J, et al. Biphasic dose response in low level light therapy - an update. Dose Response. 2011;9(4): Hawkins DH and Abrahamse H. The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation. Lasers Surg Med. 2006;38(1): Houreld NN and Abrahamse H. Laser light influences cellular viability and proliferation in diabetic-wounded fibroblast cells in a dose- and wavelengthdependent manner. Lasers Med Sci. 2008;23(1): Basso FG, Pansani TN, Turrioni AP, et al. In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts. Int J Dent. 2012;71(1): Karu T. Photobiological fundamentals of low power laser therapy. J Quant Electr. 1989;23(3):

83 25. Chen CH, Hung HS, Hsu SH. Low-energy laser irradiation increases endothelial cell proliferation, migration, and enos gene expression possibly via PI3K signal pathway. Lasers Surg Med. 2008;40(1): Huang L, Tang Y, Xing D. Activation of nuclear estrogen receptors induced by lowpower laser irradiation via PI3-K/Akt signaling cascade. J Cell Physiol. 2013;228(5): Silveira PC, Streck EL, Pinho RA. Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy. J Photochem Photobiol B. 2007;86(3): Figures Figure 1 Effect of LLLT on wound size immediately after irradiation. Linear mean length with the confidence interval for each wounded area is presented. There was no significant difference noted among groups at the beginning of the experiment (p > 0.05). Significance p <

84 Figure 2 Effect of LLLT on wound size 12 hours after irradiation. Linear mean length with the confidence interval for each wounded area is presented. There was a significant reduction in the linear mean length of the control positive (p < ) and the cells irradiated with 0.1 J/cm 2 (p = ), 0.2 J/cm 2 (p = ) and 1.2 J/cm 2 (p = 0.026) compared to the cells irradiated with 10 J/cm 2. A significant diminution of the surface area was also noted between the control positive (p < ) and the cells irradiated with the two lower energy doses (p < 0.01) compared to the control negative, and between the control positive and the cells irradiated with 1.2 J/cm 2 (p = ). There was no significant difference in the linear mean length between the control negative and the cell irradiated with 1.2 J/cm 2 (p = ), nor between the three groups with lower irradiation (p > 0.08). a,b,c,d Values with different letters are significantly (P < 0.05) different. 70

85 Figure 3 Effect of LLLT on wound size 24 hours after irradiation. Linear mean length with the confidence interval for each wounded area is presented. The linear mean length of the control positive and the cells irradiated with 0.1, 0.2, and 1.2 J/cm 2 were significantly decreased compared to the two other groups (p < ). There was also a significant difference noted in the linear mean length of the control negative compared to the cells irradiated with 10 J/cm 2 (p = ). a,b Values with different letters are significantly (P < 0.05) different. 71

86 Figure 4 Effect of LLLT on wound size 36 hours after irradiation. Linear mean length with the confidence interval for each wounded area is presented. As for the 24-hour time point results, there was no significant difference in the linear mean length between the control positive and the three lower energy density groups (p > 0.5). The linear mean length of the control positive and the cells irradiated with 0.1, 0.2, and 1.2 J/cm 2 was significantly decreased compared to the two other groups (p < 0.03). There was also a significant difference noted in the linear mean of the control negative compared to the cells irradiated with 10 J/cm 2 (p < ). significantly (P < 0.05) different. a,b Values with different letters are 72

87 Figure 5 Effect of LLLT on cellular proliferation immediately after irradiation. Mean absorbance with the confidence interval for each group is presented. There was no statistical difference noted at the beginning of the experiment (p > 0.5). Significance p <

88 Figure 6 Effect of LLLT on cellular proliferation 24 hours after irradiation. Mean absorbance with the confidence interval for each group is presented. The mean absorbance of the cells treated with 0.1, 0.2 and 1.2 J/cm 2 was significantly increased compared to the other groups (p < ). There was no significant difference between the non-irradiated cells cultured in serum-deprived medium and the cells irradiated with 10 J/cm 2 at that time point (p = ). a,b Values with different letters are significantly (P < 0.05) different. 74

89 Figure 7 Effect of LLLT on cellular proliferation 48 hours after irradiation. Mean absorbance with the confidence interval for each group is presented. The mean absorbance of the cells treated with 0.1, 0.2 and 1.2 J/cm 2 was significantly increased compared to the other groups (p <0.0001). The cells irradiated with 0.1 J/cm 2 (p = 0.01) and 0.2 J/cm 2 (p = ) had significantly greater cellular proliferation compared to the cells irradiated with 1.2 J/cm 2. There was no significant difference in the cellular proliferation rate between the two lower energy doses (p = ), nor between the control group and the cells irradiated with 10 J/cm 2 (p= ). a,b,c Values with different letters are significantly (P < 0.05) different. 75

90 3.14 Tables Table 1 Low-level laser therapy protocols for all groups for the scratch migration assay and proliferation assay Groups Medium concentration (%) Distance (cm) Spot size (cm 2 ) Time (s) Power (W) Energy density (J/cm 2 ) Control positive* Control negative Treatment Treatment Treatment Treatment *This group was not included in the proliferation assay. 76

91 CHAPTER IV An in vitro method to evaluate the effect of low-level laser therapy (LLLT) on the expression level of mirna-21 expression in a canine skin model 4.1 Introduction MicroRNAs (MiRNA or MiR) are short, non-protein coding, single stranded ribonucleic acid (RNA) molecules, that participate in the post-transcriptional control of gene expression by binding to the 3 -untranslated region (3 -UTR) of specific targeted messenger RNAs (mrnas), either inhibiting their translation or inducing their degradation [1]. Recent research has shown that many mirnas can facilitate wound healing and skin development through regulation of cellular processes, such as keratinocyte proliferation and migration [2,3]. However, their specific functions are not completely elucidated [4,5]. Of the known micrornas, mir-21 has been extensively evaluated in this project and other cellular contexts. Low-level laser therapy has been shown to have an effect on mirna expression, however, there are only three published reports supporting this finding in the literature today [6-8]. The objective of this pilot study was to determine whether mir-21 expression is affected by different doses of laser therapy, 24 hours following treatment. The mean relative mir-21 expression was analyzed and compared between non-irradiated keratinocyte cells (control group) and the cells treated with doses of 0.1, 1.2, or 10 J/cm 2. Relative mir-21 expression was normalized to snrna U6 reference gene expression. 77

92 4.2 Materials and Methods Cell culture Canine epidermal keratinocyte progenitors (CPEK) from CELLnTEC Advance Cell Systems a were cultured in 10 ml of 10% serum medium (CnT-09) in 75 cm 3 tissue culture flasks b and incubated at 37 C in an atmosphere of 5% CO 2 and 95% air. The medium was changed every forty-eight hours and replaced in a humidified incubator. For this study, CPEK cells were grown to approximately 40% confluency. Third- to fifthpassage cells were used for all experiments Study protocols Canine keratinocytes were trypsinized and plated on sterile 6-well tissue culture plates b (Corning). The cells were seeded at 1 x 10 4 cells per well in 2 ml keratinocyte growth medium CnT-09. For this experiment, four 6-well plates and three wells of one 6- well plate were used. All plates were incubated for 24 hours under the conditions previously mentioned. Following this incubation, the medium of each well was replaced with 2 ml of deprived serum (1%) medium, and incubated for an additional 12 hours. The medium of each well was then removed and replaced with 1 ml of phosphate buffered saline (PBS) c. The three wells on the half-filled 6-well plate were used for analysis of time 0 expression (control groups). These cells were collected at the time of lasertreatment using 100 µl trypsin or trypsin/edta d spun and snap frozen for subsequent RNA isolation. Each sample was collected in a 1.5 ml Eppendorf tube b and centrifuged at 1600 rpm for 2 minutes. The supernatant was removed and cells were resuspended in 0.5 ml PBS. Cells were centrifuged again at 1600 rpm for 2 minutes, the supernatant was 78

93 removed, and cells were flash frozen in liquid nitrogen. Samples were stored at -80 degrees Celsius Low-level laser therapy Laser therapy was carried out using a high-power Helium-Neon (He-Ne) Class IV laser system e. Laser safety guidelines were followed as recommended by the manufacturer. Cultured cells were irradiated using the large conical laser head provided. The handpiece was suspended from a custom-made stand holder and laser irradiation was carried at a fixed distance of 5 cm by measuring the distance between the laser head and the plate. Keratinocytes were treated in triplicate with either no laser treatment (shamirradiation), 0.1 J/cm 2, 1.2 J/cm 2, or 10 J/cm 2. The wells assigned to the sham-irradiation group were maintained under the laser head for the minimum irradiation time used in the irradiated groups, without activating the laser source. During LLLT, the non-irradiated wells were covered using a clean, double-folded, white, commercial paper towel to prevent incidental irradiation. Following laser therapy, the PBS was removed and cells were incubated in 2 ml of 1% serum media for 24 hours post-treatment at 37 C in an atmosphere of 5% CO 2 and 95% air. Cells were then collected using the collection and flash freezing methods described previously RNA isolation and microrna analysis Ribonucleic acid was isolated from each sample according to the Qiagen mirneasy Micro Kit protocol f, six to eight samples at a time. Ribonucleic acid concentrations were quantified using a Nanodrop 2000c Spectrophotometer b. 79

94 Complementary deoxyribonucleic acid (cdna) was synthesized according to the Quanta Biosciences qscript TM microrna cdna Synthesis Kit g protocol with an equal amount of starting RNA in each reaction tube. Complementary DNA was diluted in nuclease-free water to a final concentration of 1.5 ng/µl. Quantitative polymerase chain reaction (qpcr) was performed using a 3-Step Cycling Protocol with human mir-21 and snrna U6 primers. Finally, qpcr analysis of relative mir-21 expression was performed using BioRad CFX Manager software. All reagents were prepared and stored according to manufacturer s instructions. 4.3 Statistical analysis A one-way ANOVA statistical analysis was used to compare the means of relative mir-21 expression between treatment and control groups with a p-value of 0.05 accepted as statistically significant. All statistical tests were performed using Graphpad Prism 6 software h. 4.4 Results The results of this pilot study revealed no significant difference in relative mir-21 expression between untreated and laser-treated keratinocytes (p > 0.05), although there appeared to be slightly more relative mir-21 expression in cells exposed to LLLT compared to untreated controls (Figure 8). 80

95 4.5 Discussion MicroRNAs have recently been shown to play key roles in skin development, homeostasis, and are associated with many skin diseases, cancer, and wound healing processes [1-5]. The involvement and functions of mirnas in wound healing have attracted more attention recently, however, their specific role in wound healing remains largely unknown [4,5]. Although many mirnas have been implicated, mir-21 has been particularly associated with keratinocyte proliferation and migration [9,10]. Wang et al. (2012) demonstrated that mir-21 expression was increased in wound healing and that its inhibition caused significant delayed wound closure secondary to decreased collagen deposition and wound contraction [9]. In a similar study by Mills and Cowin (2013), inhibition of mir-21 has also been found to decrease the ability of fibroblasts to degrade the matrix layer in myocardial wound healing tissue [10]. These reports clearly showed that wound-induced mir-21 is crucial in wound healing, however, the exact role of mir- 21 expression in cutaneous wound healing still remains unknown. [9,10]. Low-level laser therapy has been shown to regulate mirna expression, but more research in this field is needed to make any definitive conclusions. In this pilot study, mir-21 was found to be expressed in all canine keratinocytes at the 24-hour time point, regardless of whether or not they had been laser-treated. Indeed, laser therapy had no significant effect on mir-21 expression in canine epidermal keratinocytes when compared to the untreated control. Although not significant statistically, there appeared to be slightly increased relative mir-21 expression in laser treated cells compared to the untreated control groups. Importantly, these cultures were not wounded prior to irradiation as the pilot study sought only to examine the effects of lasers directly, not 81

96 synergistic effects between wounding and laser therapy, which is a more relevant question from the biological standpoint. Furthermore, changes in mirna-21 levels may have occurred at other time points that we were unable to assess due to cost and time restrictions. Although this pilot study presents only early results on this topic, it is the first of its kind in veterinary medicine. Future research encompassing more mirnas and time points is clearly warranted to determine whether specific mirnas thought to be involved in wound healing are regulated by laser therapy, and their exact mechanism in wound healing processes. 4.6 List of abbreviations cdna CPEK MicroRNA RNA qpcr qrt-pcr Complementary deoxyribonucleic acid Canine epidermal keratinocyte progenitors mirna or mir Ribonucleic acid Quantitative polymerase chain reaction Quantitative reverse transcription polymerase chain reaction 4.7 Footnotes a. CELLnTEC Advance Cell Systems AG, Bern, Switzerland. b. Fisher Scientific, Ottawa, Ontario, Canada. c. Sigma, St. Louis, Missouri. d. Gibco-Invitrogen, Burlington, Ontario, Canada. e. CTS Therapy System, Companion Therapy Laser, LiteCure LCC, Newark, Delaware. f. Qiagen, Toronto, Ontario, Canada (Catalogue #: ). g. Quanta Biosciences, Gaithersburg, Maryland, USA (Catalogue #: ). 82

97 h. GraphPad Software Inc. La Jolla, CA, USA. 4.8 References 1. Ketting RF. MicroRNA biogenesis and function: an overview. Adv Exp Med Biol. 2011;70(4): Viticchiè G, Lena AM, Cianfarani F, et al. MicroRNA-203 contributes to skin reepithelialization. Cell Death Dis. 2012;29(3): Yang X, Wang J, Guo SL, et al. mir-21 promotes keratinocyte migration and reepithelialization during wound healing. Int J Biol Sci. 2011;7(5): Banerjee J and Sen CK. MicroRNAs in skin and wound healing. Methods Mol Biol. 2013;93(6): Banerjee J, Chan YC, Sen CK. MicroRNAs in skin and wound healing. Physiol Genomics. 2011;43(10): Kushibiki T, Hirasawa T, Okawa S, et al. Regulation of mirna Expression by Low- Level Laser Therapy (LLLT) and Photodynamic Therapy (PDT). Int J Mol Sci. 2013;14(7): Wang J, Huang W, Wu Y, et al. MicroRNA-193 pro-proliferation effects for bone mesenchymal stem cells after low-level laser irradiation treatment through inhibitor of growth family, member 5. Stem Cells Dev. 2012;21(13): Gu X, Nylander E, Coates P, et al. Effect of narrow-band ultraviolet B phototherapy on p63 and microrna (mir-21 and mir-125b) expression in psoriatic epidermis. Acta Derm Venereol. 2011;91(4): Wang T, Feng Y, Sun H, et al. mir-21 regulates skin wound healing by targeting multiple aspects of the healing process. Am J Pathol. 2012;181(6): Mills SJ and Cowin AJ. MicrorNAs and their roles in wound repair and regeneration. Wound Practice and Research: J of Aust W Manag Ass. 2013;21(1):

98 4.9 Figures Figure 8 Relative expression of mir-21 normalized to snrna U6 reference gene 24 hours following laser therapy in canine keratinocytes treated with doses of 0.1 J/cm 2, 1.2 J/cm 2, and 10 J/cm 2 compared to non-irradiated keratinocytes grown in deprived serum media. Mean relative expression with standard error of the mean (SEM) for each group is presented. There was no significant differences in mir-21 expression between groups. Significance p <

99 CHAPTER V General discussion and conclusion Wound healing is a complex, dynamic and multi-step biological process that is absolutely fundamental to the survival of complex organisms [1,2]. Keratinocytes are the primary cellular component of the epidermis and largely contribute to re-epithelialization of the injured site in wound healing [2]. Improving keratinocyte proliferation and migration may therefore be beneficial in simply accelerating the wound healing process, or in treating more challenging, chronic, non-healing lesions where proliferation may be delayed or impaired by other factors. Low-level laser therapy, also known as cold laser therapy, is the application of red and near-infrared light over injured and wounded area. It has been found to reduce pain and inflammation, and to have several other beneficial outcomes in wound healing [3]. This treatment modality is non-invasive, painless and worthy of further investigation as there is a paucity of well-conducted scientific studies supporting its use in the clinical setting [4,5]. Many authors have investigated different laser protocols in various experimental models, but there is no general consensus to fully explain the effect of laser therapy at the cellular or molecular level. Recent research has shown that mirnas have a key role in skin morphogenesis and wound healing through regulation of keratinocyte behavior [6-8]. Laser therapy has been shown in recent years to have a positive influence on the expression levels of some desirable mirnas involved in wound healing [9]. The main objective of this study was to evaluate the effect of a high-power He-Ne Class IV laser unit, comparing different doses on canine keratinocyte proliferation and 85

100 migration using an in vitro canine skin model. The second part of the project was a pilot study to perform quantitative analysis on the relative expression levels of mir-21, which is known to promote keratinocyte migration and proliferation. To the authors knowledge, this project is the first of its kind in veterinary medicine. This thesis presents novel findings using a previously-untested cell type concerning the biological effects of LLLT on important cellular parameters associated with wound healing in cultured canine epidermal keratinocytes (CPEK). The results of both the scratch migration and proliferation assays showed that canine epidermal keratinocytes produce a measurable biological response in vitro that is likely favorable for wound healing when exposed to low doses of LLLT (0.1, 0.2 or 1.2 J/cm 2), compared to non-irradiated cultures. Overall, the positive control group in which normal serum, containing numerous additional growth factors and nutrients was present, showed the fastest rate of closure, which was expected. This rate of closure was, however, not significantly faster than cells maintained in serum-deprived medium that were also exposed to any of the three lower levels of laser irradiation. For the cell proliferation studies, because the cell proliferation reagent WST-1 was added directly to the medium, a positive control group was not included as the metabolic consequences of the high serum concentration that is present alters the spectral properties of the medium, making absorbance analysis invalid between samples that contain hight serum concentration and those that do not. Furthermore, the high serum medium concentration impairs proper quantification of the formazan dye absorbance and this finding was noted while performing the pilot study; the light absorbance of the positive control group was always decreased compared to the others in all experimentations. Importantly, however, a 86

101 significant stimulatory effect of LLLT on cell proliferation of canine keratinocytes was observed compared to those grown under reduced serum conditions. It is likely that the migration assay is, to some extent, affected by the cellular proliferation occuring and the relative effects of LLLT on migration versus proliferation will need to be evaluated in future work. A clinically relevant finding of the present work is that LLLT delivered at the highest exposure dosage (10J/cm 2 ) resulted in an inhibitory effect, suggesting that dose level is extremely important in the overall biologic response. Dose-responses of this type have been previously described in several in vivo clinical experiments, animal models, and cell cultures [10-12]. The theory behind dose-response in laser therapy suggests that lower level of light may have a better effect on wound healing than a high level, which may have an inhibitory or cytotoxic effect [11,12]. The concept of biphasic doseresponse is important in LLLT and the difficulty of choosing amongst a large number of laser parameters for each treatment may explain why there is disagreement among clinical results and lack of clear conclusions regarding the reported effects [12,13]. Overall, the results of this study showed a beneficial effect of LLLT in this canine in vitro wound healing model and helped to identify the individual processes which are influenced in keratinocytes. Low-level laser therapy may stimulate or impede cellular processes in a dose-dependent manner, suggesting that appropriate protocol selection will be critical for improved clinical outcomes [14]. Despite the fact that this study included a large sample size and produced statistically significant results, there are clear limitations worth mentioning. First, the exact dosage of laser irradiation received by the exposed canine keratinocyte cells is 87

102 unknown; a laser power meter was not used to calibrate the power output of the laser unit at the beginning of the experiment, and the power loss through the acrylic plate was not determined. In their study, Basso and colleagues (2010) obtained a 5% power loss from the plate using a potentiometer, which they reported being minimal [13]. In the present study, the laser system unit provided was modern and was calibrated prior to the experiments; the results obtained with the selected laser parameters throughout the study period were therefore likely accurate. Although the results of the study showed a significant benefit of LLLT in terms of migration, they are limited to the specific laser parameters selected here and it cannot be applied directly to a veterinary clinical settings. No direct extrapolation to in vivo wound healing can be made. Large, well-designed, prospective clinical trials are needed to fully evaluate the safety and efficacy of LLLT in veterinary medicine and to determine whether similar biphasic results occur in an in vivo context. In the second part of the project, a pilot study was performed to identify whether mir-21 expression would be increased in cultured canine epidermal keratinoctyes exposed to the the same laser doses as used in the scratch migration and proliferation assays, using standard qrt-pcr analysis for micrornas. The results indicated that there was no significant difference in relative mir-21 expression between untreated and lasertreated keratinocytes, but there appeared to be slightly higher mir-21 expression in cells exposed to LLLT compared to untreated controls. Although this part of the project is only a pilot study at this point and that no clear conclusion can be made, these findings support the rationale for additional studies designed to determine the mechanism of action of LLLT at the cellular level. Further investigation is needed to fully elucidate the roles, 88

103 if any, of mirnas in normal skin, their regulation in wound healing and the effect of laser therapy on this complex regulation. This pilot study has provided some of the first evidence that the effect of laser therapy on canine epidermal keratinocyte cells may be associated, at least in part, with increased expression levels of mir-21. Further well designed research is warranted to further evaluate the relationship between laser therapy and mirna expression. Other mirnas such as mirna-203, which has also been found to be important in keratinocyte activity during wound healing, should also be evaluated [15]. In conclusion, LLLT may be an attractive tool for the healing of burns, acute and chronic non-healing wounds, as well as ulcers of different etiologies [16]. By decreasing healing time, the biological effect of this therapy may also significantly reduce the risk of infection and other complications, as well as the costs associated with their intensive management [16,17]. It is clearly the responsibility of veterinarians involved in research to substantiate or refute the reported beneficial effects associated with the use of LLLT in our companion animal patients. Further in vitro and in vivo studies are required to fully investigate the effects of LLLT on canine wound healing and the mechanisms by which this treatment mediates its effects so that sound and scientifically-based recommendations can be made. 5.1 References 1. Singer AJ and Clark RA. Cutaneous wound healing. N Engl J Med. 1999;341(10):

104 2. Velnar T, Bailey T, Smrkolj V. The wound healing process: an overview of the cellular and molecular mechanisms. J Int Med Res. 2009;37(5): Medrado AR, Pugliese LS, Reis SR, et al. Influence of low level laser therapy on wound healing and its biological action upon myofibroblasts. Lasers Surg Med. 2003;32(3): Hawkins D, Houreld N, Abrahamse H. Low-level laser therapy (LLLT) as an effective therapeutic modality for delayed wound healing. Ann N Y Acad Sci. 2005;1056(1): Prindeze NJ, Moffatt LT, Shupp JW. Mechanisms of action for light therapy: a review of molecular interactions. Exp Biol Med (Maywood). 2012;237(11): Banerjee J, Chan YC, Sen CK. MicroRNAs in skin and wound healing. Physiol Genomics. 2011;43(10): Banerjee J and Sen CK. MicroRNAs in skin and wound healing. Methods Mol Biol. 2013;93(6): Yi R, O'Carroll D, Pasolli HA, et al. Morphogenesis in skin is governed by discrete sets of differentially expressed micrornas. Nat Genet. 2006;38(3): Kushibiki T, Hirasawa T, Okawa S, et al. Regulation of mirna Expression by Low- Level Laser Therapy (LLLT) and Photodynamic Therapy (PDT). Int J Mol Sci. 2013;14(7): Demidova-Rice TN, Salomatina EV, Yaroslavsky AN, et al. Low-level light stimulates excisional wound healing in mice. Lasers Surg Med. 2007;39(9): Huang YY, Chen AC, Carroll JD, et al. Biphasic dose response in low level light therapy. Dose Response. 2009;7(4): Huang YY, Sharma SK, Carroll J, et al. Biphasic dose response in low level light therapy - an update. Dose Response. 2011;9(4): Basso FG, Pansani TN, Turrioni AP, et al. In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts. Int J Dent. 2012;71(1): Hawkins DH and Abrahamse H. The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation. Lasers Surg Med. 2006;38(1): Lai WF and Siu PM. MicroRNAs as regulators of cutaneous wound healing. J Biosci. 2014;39(3):

105 16. de Jesus Guirro RR, de Oliveira Guirro EC, Martins CC, et al. Analysis of lowlevel laser radiation transmission in occlusive dressings. Photomed Laser Surg. 2010;28(4): Saltmarche AE. Low-level laser therapy for healing acute and chronic wounds - the extendicare experience. Int Wound J. 2008;5(2):

106 CHAPTER VI Appendices 6.1- Figures A. 1 x 10 6 cells/well B. 4 x 10 4 cells/well Figure 9 Photographs representing the different cellular concentration of canine epidermal keratinocytes seeded in 6-well plates for the (A) scrath migration assay and for the (B) proliferation assay. Images were obtained from a motorized inverted microscope. 92

107 Figure 10 Photograph demonstrating a scratch created in a canine epidermal keratinocyte cell monolayer for the sracth migration assay, at the beginning of the experiment. Images were obtained from a motorized inverted microscope. 93

108 Control positive Control negative Treatment 1 Treatment 4 Figure 11 Representative scratch migration assay in canine epidermal keratinocyte cells at the 12-hour time point. Groups presented in this figure include non-irradiated cells maintained in rich serum medium (control positive), non-irradiated cells maintained in starved serum medium (control negative), cells irradiated with 0.1 J/cm2 (treatment 1), and cells irradiated with 10 J/cm2, both also maintained in starved serum medium. The scratch of the control positive and treatment 1 groups are completely closed, whereas the scratch of the control negative and treatment 4 are still clearly visible at that time. Images were obtained from a motorized inverted microscope. 94

109 Figure 12 Photograph of the high-power Helium-Neon (He-Ne) Class IV laser system unit suspended with a custom-made stand holder. 95